Synthesis of epothilones, intermediates thereto, analogues and uses thereof

ABSTRACT

The present invention provides convergent processes for preparing epothilone A and B, desoxyepothilones A and B, and analogues thereof. Also provided are analogues related to epothilone A and B and intermediates useful for preparing same. The present invention further provides novel compositions based on analogues of the epothilones and methods for the treatment of cancer and cancer which has developed a multidrug-resistant phenotype.

[0001] This application is based on U.S. Provisional Applications SerialNos. 60/032,282, 60/033,767, 60/047,566, 60/047,941, and 60/055,533,filed Dec. 3, 1996, Jan. 14, 1997, May 22, 1997, May 29, 1997, and Aug.13, 1997, respectively, the contents of which are hereby incorporated byreference into this application. This invention was made with governmentsupport under grants CA-28824, CA-39821, CA-GM 72231, CA-62948, andAI0-9355 from the National Institutes of Health, and grant CHE-9504805from the National Science Foundation.

FIELD OF THE INVENTION

[0002] The present invention is in the field of epothilone macrolides.In particular, the present invention relates to processes for thepreparation of epothilones A and B, desoxyepothilones A and B, andanalogues thereof which are useful as highly specific, non-toxicanticancer therapeutics. In addition, the invention provides methods ofinhibiting multidrug resistant cells. The present invention alsoprovides novel compositions of matter which serve as intermediates forpreparing the epothilones.

[0003] Throughout this application, various publications are referredto, each of which is hereby incorporated by reference in its entiretyinto this application to more fully describe the state of the art towhich the invention pertains.

BACKGROUND OF THE INVENTION

[0004] Epothilones A and B are highly active anticancer compoundsisolated from the Myxobacteria of the genus Sorangium. The fullstructures of these compounds, arising from an x-ray crystallographicanalysis were determined by Höfle. G. Höfle et al., Angew. Chem. Int.Ed. Engl., 1996, 35, 1567. The total synthesis of the epothilones is animportant goal for several reasons. Taxol is already a useful resourcein chemotherapy against ovarian and breast cancer and its range ofclinical applicability is expanding. G. I. Georg et al., TaxaneAnticancer Agents; American Cancer Society: San Diego, 1995. Themechanism of the cytotoxic action of taxol, at least at the in vitrolevel, involves stabilization of microtubule assemblies. P. B. Schiff etal., Nature (London), 1979, 277, 665. A series of complementary in vitroinvestigations with the epothilones indicated that they share themechanistic theme of the taxoids, possibly down to the binding sites totheir protein target. D. M. Bollag et al, Cancer Res., 1995, 55, 2325.Moreover, the epothilones surpass taxol in terms of cytotoxicity and farsurpass it in terms of in vitro efficacy against drug resistant cells.Since multiple drug resistance (MDR) is one of the serious limitationsof taxol (L. M. Landino and T. L. MacDonald in The Chemistry andPharmacology of Taxol and its Derivatives, V. Farin, Ed., Elsevier: NewYork, 1995, ch. 7, p. 301), any agent which promises relief from thisproblem merits serious attention. Furthermore, formulating theepothilones for clinical use is more straightforward than taxol.

[0005] Accordingly, the present inventors undertook the total synthesisof the epothilones, and as a result, have developed efficient processesfor synthesizing epothilones A and B, the correspondingdesoxyepothilones, as well as analogues thereof. The present inventionalso provides novel intermediates useful in the synthesis of epothilonesA and B and analogues thereof, compositions derived from suchepothilones and analogues, purified compounds of epothilones A and B,and desoxyepothilones A and B, in addition to methods of use of theepothilone analogues in the treatment of cancer. Unexpectedly, certainepothilones have been found to be effective not only in reversingmulti-drug resistance in cancer cells, both in vitro and in vivo, buthave been determined to be active as collateral sensitive agents, whichare more cytotoxic towards MDR cells than normal cells, and assynergistic agents, which are more active in combination with othercytotoxic agents, such as vinblastin, than the individual drugs would bealone at the same concentrations. Remarkably, the desoxyepothilones ofthe invention have exceptionally high specificity as tumor cytotoxicagents in vivo, more effective and less toxic to normal cells than theprincipal chemotherapeutics currently in use, including taxol,vinblastin, adriamycin and camptothecin.

SUMMARY OF THE INVENTION

[0006] One object of the present invention is to provide processes forthe preparation of epothilones A and B, and desoxyepothilones A and B,and related compounds useful as anticancer therapeutics. Another objectof the present invention is to provide various compounds useful asintermediates in the preparation of epothilones A and B as well asanalogues thereof.

[0007] A further object of the present invention is to provide syntheticmethods for preparing such intermediates. An additional object of theinvention is to provide compositions useful in the treatment of subjectssuffering from cancer comprising any of the analogues of the epothilonesavailable through the preparative methods of the invention optionally incombination with pharmaceutical carriers.

[0008] A further object of the invention is to provide methods oftreating subjects suffering from cancer using any of the analogues ofthe epothilones available through the preparative methods of theinvention optionally in combination with pharmaceutical carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1(A) shows a retrosynthetic analysis for epothilone A and B.

[0010]FIG. 1(B) provides synthesis of compound 11. (a) t-BuMe₂OTf,2,6-lutidine, CH₂Cl₂, 98%; (b) (1) DDQ, CH₂Cl₂/H₂O, 89%; (2) (COCl)₂,DMSO, CH₂Cl₂, −78° C.; then Et₃N, −78° C.

rt, 90%; (c) MeOCH₂PPh₃Cl, t-BuOK, THF, 0° C.

rt, 86%; (d) (1) p-TsOH, dioxane/H₂O, 50° C., 99%; (2) CH₃PPh₃Br,NaHMDS, PhCH₃, 0° C.

rt, 76%; (e) Phl(OCOCF₃)₂, MeOH/THF, rt, 0.25 h, 92%.

[0011]FIG. 2 provides key intermediates in the preparation of 12,13-E-and -Z-deoxyepothilones.

[0012]FIG. 3(A) provides syntheses of key iodinated intermediates usedto prepare hydroxymethylene- and hydroxypropylene-substituted epothilonederivatives.

[0013]FIG. 3(B) provides methods of preparing hydroxymethylene- andhydroxypropylene-substituted epothilone derivatives, said methods beinguseful generally to prepare 12,1 3-E epothilones wherein R is methyl,ethyl, n-propyl, and n-hexyl from the corresponding E-vinyl iodides.

[0014]FIG. 3(B) shows reactions leading to benzoylatedhydroxymethyl-substituted desoxyepothilone andhydroxymethylene-substituted epothilone (epoxide).

[0015]FIG. 4(A) provides synthesis of compound 19. (a) DHP, PPTS,CH₂Cl₂, rt: (b) (1) Me₃SiCCLi, BF₃.OEt₂, THF, −78° C.; (2) MOMCl,I-Pr₂NEt, Cl(CH₂)₂Cl, 55° C.; (3) PPTS, MeOH, rt; (c) (1) (COCl)₂, DMSO,CH₂Cl₂, −78° C.; then Et₃N, −78° C.

rt; (2) MeMgBr, Et₂O, 0° C.

rt, (3) TPAP NMO, 4 Å mol. sieves, CH₂Cl₂, 0° C.

rt; (d) 16, n-BuLi, THF, −78° C.; then 15, THF, −78° C.

rt; (e) (1) N-iodosuccinimide, AgNO₃, (CH₃)₂CO; (2) Cy₂BH, Et₂O, AcOH;(f) (1) PhSH, BF₃.OEt₂, CH₂Cl₂, rt; (2) Ac₂O, pyridine, 4-DMAP, CH₂Cl₂,rt.

[0016]FIG. 4(B) presents synthesis of compound 1. (a) 11, 9-BBN, THF,rt; then PdCl₂(dppf)₂, Cs₂CO₃, Ph₃As, H₂O, DMF, 19, rt, 71%; (b) p-TsOH,dioxane/H₂O, 50° C.; (c) KHMDS, THF, −78° C., 51%; (d) (1) HF-pyridine,pyridine, THF, rt, 97%; (2) t-BuMe₂ SiOTf, 2,6-lutidine, CH₂Cl₂, −25°C., 93%; (3) Dess-Martin periodinane, CH₂Cl₂, 87%; (4) HF•pyridine, THF,rt, 99%; (e) dimethyldioxirane, CH₂Cl₂, 0.5 h, −50° C., 45% (≧20:1).

[0017]FIG. 5 shows a scheme of the synthesis of the “left wing” ofepothilone A.

[0018]FIG. 6 provides a scheme of an olefin metathesis route toepothilone A and other analogues.

[0019]FIG. 7 illustrates a convergent strategy for a total synthesis ofepothilone A (1) and the glycal cyclopropane solvolysis strategy for theintroduction of geminal methyl groups.

[0020]FIG. 8 provides an enantioselective synthesis of compound 15B.

[0021]FIG. 9 shows the construction of epothilone model systems 20B,21B, and 22B by ring-closing olefin metathesis.

[0022]FIG. 10 illustrates a sedimentation test for natural, syntheticand desoxyepothilone A.

[0023]FIG. 11 illustrates a sedimentation test for natural, syntheticand desoxyepothilone A after cold treatment at 4° C.

[0024]FIG. 12 illustrates (A) structures of epothilones A (1) and B (2)and (B) of Taxol™ (1A).

[0025]FIG. 13 shows a method of elaborating acyclic stereochemicalrelationships based on dihydropyrone matrices.

[0026]FIG. 14 shows the preparation of intermediate 4A.

[0027]FIG. 15 shows an alternative enantioselective synthesis ofcompound 17A.

[0028]FIG. 16 provides a synthetic pathway to intermediate 13C. (a) 1.tributyl allyltin, (S)-(−)-BINOL, Ti(Oi-Pr)₄, CH₂Cl₂, −20° C., 60%, >95%e.e.; 2. Ac₂O, Et₃N, DMAP, CH₂Cl₂, 95%; (b) 1. OsO₄, NMO, acetone/H₂O,0° C.; 2. NalO₄, THF/H₂O; (c) 12, THF, −20° C., Z isomer only, 25% from10; (d) Pd(dppf)₂, Cs₂CO₃, Ph₃As, H₂O, DMF, rt. 77%.

[0029]FIG. 17 provides a synthetic pathway to intermediate epothilone B(2). (a) p-TsOH, dioxane/H₂O, 55° C., 71%; (b) KHMDS, THF, −78° C., 67%,α/β: 1.5:1; (c) Dess-Martin periodinane, CH₂Cl₂; (d) NaBH₄, MeOH, 67%for two steps; (e) 1. HF•pyridine, pyridine, THF, rt, 93%; 2. TBSOTf,2,6-lutidine, CH₂Cl₂, −30° C., 89%; 3. Dess-Martin periodinan CH₂Cl₂,67%; (f) HF•pyridine, THF, rt, 80%; (g) dimethyldioxirane, CH₂Cl₂, −50°C., 70%.

[0030]FIG. 18 provides a synthetic pathway to a protected intermediatefor 8-desmethyl deoxyepothilone A.

[0031]FIG. 19 provides a synthetic pathway to 8-desmethyldeoxyepothilone A, and structures of trans-8-desmethyl-desoxyepothioloneA and a trans-iodoolefin intermediate thereto.

[0032]FIG. 20 shows (top) structures of epothilones A and B and8-desmethylepothilone and (bottom) a synthetic pathway to intermediateTBS ester 10 used in the preparation of desmethylepothilone A. (a)(Z)-Crotyl-B[(−)-lpc]₂, −78° C., Et₂O, then 3N NaOH, 30% H₂O₂; (b)TBSOTf, 2,6-lutidine, CH₂Cl₂ (74% for two steps, 87% ee); (c) O₃,CH₂Cl₂/MeOH, −78° C., then DMS, (82%); (d) t-butyl isobutyrylacetate,NaH, BuLi, 0° C., then 6 (60%, 10:1); (e) Me₄NBH(OAc)₃, −10° C. (50%,10:1 α/β) or NaBH₄, MeOH, THF, 0° C., (88%, 1:1 α/β); (f) TBSOTf,2,6-lutidine, −40° C., (88%); (g) Dess-Martin periodinane, (90%); (h)Pd(OH)₂, H₂, EtOH (96%); (I) DMSO, oxalyl chloride; CH₂Cl₂, −78° C.(78%); (j) Methyl triphenylphosphonium bromide, NaHMDS, THF, 0° C.(85%); (k) TBSOTf, 2,6-lutidine, CH₂Cl₂, rt (87%).

[0033]FIG. 21 shows a synthetic pathway to 8-desmethylepothilone A. (a)Pd(dppf)₂Cl₂, Ph₃As, Cs₂CO₃, H₂O, DMF, rt (62%); (b) K₂CO₃, MeOH, H₂O(78%); (c) DCC, 4-DMAP, 4-DMAP•HCl, CHCl₃ (78%); (d) HF•pyr, THF, rt(82%), (e) 3,3-dimethyl dioxirane, CH₂Cl₂, −35° C. (72%, 1.5:1).

[0034]FIG. 22 shows a synthetic pathway to prepare epothilone analogue27D.

[0035]FIG. 23 shows a synthetic pathway to prepare epothilone analogue24D.

[0036]FIG. 24 shows a synthetic pathway to prepare epothilone analogue19D.

[0037]FIG. 25 shows a synthetic pathway to prepare epothilone analogue20D.

[0038]FIG. 26 shows a synthetic pathway to prepare epothilone analogue22D.

[0039]FIG. 27 shows a synthetic pathway to prepare epothi lone analogue12-hydroxy ethyl-epothilone.

[0040]FIG. 28 shows the activity of epothilone analogues in asedimentation test in comparison with DMSO, epothilone A and/or B.Structures 17-20, 22, and 24-27 are shown in FIGS. 29-37, respectively.Compounds were added to tubulin (1 mg/ml) to a concentration of 10 μM.The quantity of microtubules formed with epothilone A was defined as100%.

[0041]FIG. 29 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #17.

[0042]FIG. 30 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #18.

[0043]FIG. 31 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #19.

[0044]FIG. 32 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #20.

[0045]FIG. 33 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #22.

[0046]FIG. 34 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #24.

[0047]FIG. 35 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #25.

[0048]FIG. 36 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #26.

[0049]FIG. 37 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #27.

[0050]FIG. 38 provides a graphical representation of the effect offractional combinations of cytotoxic agents.

[0051]FIG. 39 shows epothilone A and epothilone analogues #1-7.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

[0052]FIG. 40 shows epothilone B and epothilone analogues #8-16.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

[0053]FIG. 41 shows epothilone analogues #17-25. Potencies against humanleukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR (resistant) sublinesare shown in round and square brackets, respectively.

[0054]FIG. 42(A) shows epothilone analogues #26-34. Potencies againsthuman leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR (resistant)sublines are shown in round and square brackets, respectively.

[0055]FIG. 42(B) shows epothilone analogues #35-46. Potencies againsthuman leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR (resistant)sublines are shown in round and square brackets, respectively.

[0056]FIG. 42(C) shows epothilone analogues #47-49.

[0057]FIG. 43(A) shows antitumor activity of desoxyepothilone B againstMDR MCF-7/Adr xenograft in comparison with taxol. Control (♦);desoxyepothilone B (▪; 35 mg/kg); taxol (▴; 6 mg/kg); adriamycin (x;1.8mg/kg); i.p. Q2Dx5; start on day 8.

[0058]FIG. 43(B) shows antitumor activity of epothilone B against MDRMCF-7/Adr xenograft in comparison with taxol. Control (♦); epothilone B(▪; 25 mg/kg; non-toxic dose); taxol (▴; 6 mg/kg; half LD₅₀); adriamycin(x;1.8 mg/kg); i.p. Q2Dx5; start on day 8.

[0059]FIG. 44(A) shows toxicity of desoxyepothilone B in B6D2F₁ micebearing B16 melanoma. Body weight was determined at 0, 2,4, 6, 8, 10 and12 days. Control (▴); desoxyepothilone B (∘; 10 mg/kg QDx8; 0 of 8died); desoxyepothilone B (; 20 mg/kg QDx6; 0 of 8 died). Injectionswere started on day 1.

[0060]FIG. 44(B) shows toxicity of epothilone B in B6D2F₁ mice bearingB16 melanoma. Body weight was determined at 0, 2,4, 6, 8,10 and 12 days.Control (▴); epothilone B (∘; 0.4 mg/kg QDx6; 1 of 8 died of toxicity);epothilone B (; 0.8 mg/kg QDx5; 5 of 8 died). Injections were startedon day 1.

[0061]FIG. 45(A) shows comparative therapeutic effect ofdesoxyepothilone B and taxol on nude mice bearing MX-1 xenoplant. Tumor,s.c.; drug administered i.p., Q2Dx5, start on day 7. control (♦); Taxol(□; 5 mg/kg, one half of LD₅₀); desoxyepothilone B (Δ; 25 mg/kg;nontoxic dose).

[0062]FIG. 45(B) shows comparative therapeutic effect ofdesoxyepothilone B and taxol on nude mice bearing MX-1 xenoplant. Tumor,s.c.; drug administered i.p., Q2Dx5, start on day 7. control (♦); Taxol(□; 5 mg/kg, one half of LD₅₀, given on days 7, 9, 11, 13, 15; then 6mg/kg, given on days 17. 19, 23, 24, 25); desoxyepothilone B (n=3; Δ, x,*; 25 mg/kg, nontoxic dose, given to three mice on days 7, 9, 11, 13,15; then 35 mg/kg, given on days 17. 19, 23, 24, 25).

[0063]FIG. 46 shows the effect of treatment with desoxyepothilone B (35mg/kg), taxol (5 mg/kg) and adriamycin (2 mg/kg) of nude mice bearinghuman MX-1 xenograft on tumor size between 8 and 18 days afterimplantation. Desoxyepothilone B (□), taxol (Δ), adriamycin (X), control(♦); i.p. treatments were given on day 8, 10, 12, 14 and 16.

[0064]FIG. 47 shows the relative toxicity of epothilone B (□; 0.6 mg/kgQDx4; i.p.) and desoxyepothilone B (Δ; 25 mg/kg QDx4; i.p.) versuscontrol (♦) in normal nude mice. Body weight of mice was determineddaily after injection. For epothilone B, 8 of 8 mice died of toxicity ondays 5, 6, 6, 7, 7, 7, 7, and 7; for desoxyepothilone B, all six micesurvived.

[0065]FIG. 48 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #43.

[0066]FIG. 49 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #45.

[0067]FIG. 50 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #46.

[0068]FIG. 51 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #47.

[0069]FIG. 52 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #48.

DETAILED DESCRIPTION OF THE INVENTION

[0070] As used herein, the term “linear or branched chain alkyl”encompasses, but is not limited to, methyl, ethyl, propyl, isopropyl,t-butyl, sec-butyl, cyclopentyl or cyclohexyl.

[0071] The alkyl group may contain one carbon atom or as many asfourteen carbon atoms, but preferably contains one carbon atom or asmany as nine carbon atoms, and may be substituted by various groups,which include, but are not limited to, acyl, aryl, alkoxy, aryloxy,carboxy, hydroxy, carboxamido and/or N-acylamino moieties.

[0072] As used herein, the terms “alkoxycarbonyl”, “acyl” and “alkoxy”encompass, but are not limited to, methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, n-butoxycarbonyl, benzyloxycarbonyl,hydroxypropylcarbonyl, aminoethoxycarbonyl, sec-butoxycarbonyl andcyclopentyloxycarbonyl. Examples of acyl groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl and penanoyl. Examples ofalkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy,n-butoxy, sec-butoxy and cyclopentyloxy.

[0073] As used herein, an “aryl” encompasses, but is not limited to, aphenyl, pyridyl, pyrryl, indolyl, naphthyl, thiophenyl or furyl group,each of which may be substituted by various groups, which include, butare not limited, acyl, aryl alkoxy, aryloxy, carboxy, hydroxy,carboxamido or N-acylamino moieties. Examples of aryloxy groups include,but are not limited to, a phenoxy, 2-methylphenoxy, 3-methylphenoxy and2-naphthoxy. Examples of acyloxy groups include, but are not limited to,acetoxy, propanoyloxy, butyryloxy, pentanoyloxy and hexanoyloxy.

[0074] The subject invention provides chemotherapeutic analogues ofepothilone A and B, including a compound having the structure:

[0075] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or 3. In oneembodiment, the invention provides the compound having the structure:

[0076] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH,or (CH₂)₃OH.

[0077] The invention also provides a compound having the structure:

[0078] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄, and R₅, are independently H ora linear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. In acertain embodiment, the invention provides a compound having thestructure:

[0079] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl orCH₂OH.

[0080] In addition, the invention provides a compound having thestructure:

[0081] wherein R, R₀ and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. Inparticular, the invention provides a compound having the structure:

[0082] wherein R is H, methyl, ethyl, n-propyl, n-butyl, CH₂OH or(CH₂)₃OH.

[0083] The invention further provides a compound having the structure:

[0084] wherein R, R₀ and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2 or 3.

[0085] The invention also provides a compound having the structure:

[0086] The subject invention also provides various intermediates usefulfor the preparation of the chemotherapeutic compounds epithilone A andB, as well as analogues thereof. Accordingly, the invention provides akey intermediate to epothilone A and its analogues having the structure:

[0087] wherein R is hydrogen, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; wherein R′ is hydrogen, methyl, ethyl,n-propyl, n-hexyl,

[0088] CH₂OTBS or (CH₂)₃-OTBDPS; and X is a halide. In one embodiment,the subject invention provides a compound of the above structure whereinR is acetyl and X is iodo.

[0089] The subject invention also provides an intermediate having thestructure:

[0090] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR)₂, (SR)₂, —(O—(CH₂) _(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; and wherein n is 2, 3 or 4.

[0091] wherein R is H or methyl.

[0092] Another analogue provided by the invention has the structure:

[0093] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH,or (CH₂)₃OH.

[0094] Additionally, the subject invention provides an analogue havingthe structure:

[0095] wherein R is H or methyl. The scope of the present inventionincludes compounds wherein the C₃ carbon therein possesses either an Ror S absolute configuration, as well as mixtures thereof.

[0096] The subject invention further provides an analogue of epothiloneA having the structure:

[0097] The subject invention also provides synthetic routes to preparethe intermediates for preparing epothilones. Accordingly, the inventionprovides a method of preparing a Z-iodoalkene ester having thestructure:

[0098] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises (a)coupling a compound having the structure:

[0099] with a methyl ketone having the structure:

[0100] wherein R′ and R″ are independently a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryl or benzyl, under suitableconditions to form a compound having the structure:

[0101] (b) treating the compound formed in step (a) under suitableconditions to form a Z-iodoalkene having the structure:

[0102] and (c) deprotecting and acylating the Z-iodoalkene formed instep (b) under suitable conditions to form the Z-iodoalkene ester. Thecoupling in step (a) may be effected using a strong base such as n-BuLiin an inert polar solvent such as tetrahydrofuran (THF) at lowtemperatures, typically below −50° C., and preferably at −78° C. Thetreatment in step (b) may comprise sequential reaction withN-iodosuccinimide in the presence of Ag(I), such as silver nitrate, in apolar organic solvent such as acetone, followed by reduction conditions,typically using a hydroborating reagent, preferably using Cy₂BH.Deprotecting step (c) involves contact with a thiol such as thiophenolin the presence of a Lewis acid catalyst, such as borontrifluoride-etherate in an inert organic solvent such asdichloromethane, followed by acylation with an acyl halide, such asacetyl chloride, or an acyl anhydride, such as acetic anhydride in thepresence of a mild base such as pyridine and/or 4-dimethyaminopyridine(DMAP) in an inert organic solvent such as dichloromethane.

[0103] The subject invention also provides a method of preparing aZ-haloalkene ester having the structure:

[0104] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; and wherein X is ahalogen, which comprises (a) oxidatively cleaving a compound having thestructure:

[0105] under suitable conditions to form an aldehyde intermediate; and(b) condensing the aldehyde intermediate with a halomethylene transferagent under suitable conditions to form the Z-haloalkene ester. In oneembodiment of the method, X is iodine. In another embodiment, the methodis practiced wherein the halomethylene transfer agent is Ph₃P═CHI or(Ph₃P⁺CH₂I)I⁻. Disubstituted olefins may be prepared using thehaloalkylidene transfer agent Ph₃P═CR′I, wherein R′ is hydrogen, methyl,ethyl, n-prop-yl, n-hexyl,

[0106] CO₂Et or (CH₂)₃OTBDPS. The oxidative step (a) can be performedusing a mild oxidant such as osmium tetraoxide at temperatures of about0° C., followed by treatment with sodium periodate, or with leadtetraacetate/sodium carbonate, to complete the cleavage of the terminalolefin, and provide a terminal aldehyde. Condensing step (b) occurseffectively with a variety of halomethylenating reagents, such as Wittigreagents.

[0107] The subject invention further provides a method of preparing anoptically pure compound having the structure:

[0108] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)condensing an allylic organometallic reagent with an unsaturatedaldehyde having the structure:

[0109] under suitable conditions to form an alcohol, and, optionallyconcurrently therewith, optically resolving the alcohol to form anoptically pure alcohol having the structure:

[0110] (b) alkylating or acylating the optically pure alcohol formed instep (a) under suitable conditions to form the optically pure compound.In one embodiment of the method, the allylic organometallic reagent isan allyl(trialkyl)stannane. In another embodiment, the condensing stepis effected using a reagent comprising a titanium tetraalkoxide and anoptically active catalyst. In step (a) the 1,2-addition to theunsaturated aldehyde may be performed using a variety of allylicorganometallic reagents, typically with an allyltrialkylstannane, andpreferably with allyltri-n-butylstannane, in the presence of chiralcatalyst and molecular sieves in an inert organic solvent such asdichloromethane. Preferably, the method may be practiced using titaniumtetraalkoxides, such as titanium tetra-n-propoxide, and S-(−)BINOL asthe optically active catalyst. Alkylating or acylating step (b) iseffected using any typical alkylating agent, such as alkylhalide oralkyl tosylate, alkyl triflate or alkyl mesylate, any typical acylatingagent, such as acetyl chloride, acetic anhydride, benzoyl chloride orbenzoyl anhydride, in the presence of a mild base catalyst in an inertorganic solvent, such as dichloromethane.

[0111] The subject invention also provides a method of preparing anopen-chain aldehyde having the structure:

[0112] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, which comprises:(a) cross-coupling a haloolefin having the structure:

[0113] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, and X is a halogen, with a terminalolefin having the structure:

[0114] wherein (OR′″)₂ is (OR₀)₂, (SR₀)₂, —(O—(CH₂)_(n)—O)—,—(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)— where R₀ is a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl; and wherein n is 2,3 or 4, under suitable conditions to form a cross-coupled compoundhaving the structure:

[0115] wherein Y is CH(OR*)₂ where R* is a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryloxyalkyl; and (b)deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain compound. Cross-coupling step(a) is effected using reagents known in the art which are suited to thepurpose. For example, the process may be carried out by hydroboratingthe pre-acyl component with 9-BBN. The resulting mixed borane may thenbe cross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂,or any known equivalent thereof, in the presence of such ancillaryreagents as cesium carbonate and triphenylarsine. Deprotecting step (b)can be carried out with a mild acid catalyst such as p-tosic acid, andtypically in a mixed aqueous organic solvent system, such asdioxane-water. The open-chain compound can be cyclized using any of avariety of non-nucleophilic bases, such as potassiumhexamethyldisilazide or lithium diethyamide.

[0116] The subject invention also provides a method of preparing anepothilone having the structure:

[0117] which comprises: (a) deprotecting a cyclized compound having thestructure:

[0118] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, under suitableconditions to form a deprotected cyclized compound and oxidizing thedeprotected cyclized compound under suitable conditions to form adesoxyepothilone having the structure:

[0119] and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone. Deprotecting step (a) iseffected using a sequence of treatments comprising a catalyst such asHF-pyridine, followed by t-butyldimethylsilyl triflate in the presenceof a base such as lutidine. Dess-Martin oxidation and furtherdeprotection with a catalyst such as HF-pyridine provides thedesoxyepothilone. The latter compound can then be epoxidized in step (b)using any of a variety of epoxidizing agents, such acetic peracid,hydrogen peroxide, perbenzoic acid, m-chloroperbenzoic acid, butpreferably with dimethyldioxirane, in an inert organic solvent such asdichloromethane.

[0120] The subject invention further provides a method of preparing anepothilone precursor having the structure:

[0121] wherein R₁ is hydrogen or methyl; wherein X is O, or a hydrogenand OR″, each singly bonded to carbon; and wherein R₀, R′ and R″ areindependently hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises (a) coupling a compoundhaving the structure:

[0122] wherein R is an acetyl, with an aldehyde having the structure:

[0123] wherein Y is oxygen, under suitable conditions to form an aldolintermediate and optionally protecting the aldol intermediate undersuitable conditions to form an acyclic epthilone precursor having thestructure:

[0124] (b) subjecting the acylic epothilone precursor to conditionsleading to intramolecular olefin metathesis to form the epothiloneprecursor. In one embodiment of the method, the conditions leading tointramolecular olefin metathesis require the presence of anorganometallic catalyst. In a certain specific embodiment of the method,the catalyst contains Ru or Mo. The coupling step (a) may be effectedusing a nonnucleophilic base such as lithium diethylamide or lithiumdiisopropylamide at subambient temperatures, but preferably at about−78° C. The olefin metathesis in step (b) may be carried out using anycatalyst known in the art suited for the purpose, though preferablyusing one of Grubbs's catalysts.

[0125] In addition, the present invention provides a compound useful asan intermediate for preparing epothilones having the structure:

[0126] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR*)₂, (SR*)₂, —(O—(CH₂) _(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; wherein R* is a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl; wherein R₂B is a linear, branched orcyclic boranyl moiety; and wherein n is 2, 3 or 4. In certainembodiments, the invention provides the compound wherein R′ is TBS, R″is TPS and X is (OMe)₂. A preferred example of R₂B is derived from9-BBN.

[0127] The invention also provides the compound having the structure:

[0128] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyi, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR)₂, (SR)₂, —(O—(CH₂) _(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; and wherein n is 2, 3 or 4. In certain embodiments,the invention provides the compound wherein R′ is TBS, R″ is TPS and Xis (OMe)₂.

[0129] The invention further provides a desmethylepothilone analogouehaving the structure:

[0130] wherein R is H or methyl.

[0131] The invention provides a compound having the structure:

[0132] wherein R is H or methyl.

[0133] The invention also provides a trans-desmethyldeoxyepothiloneanalogue having the structure:

[0134] wherein R is H or methyl.

[0135] The invention also provides a trans-epothilone having thestructure:

[0136] wherein R is H or methyl.

[0137] The invention also provides a compound having the structure:

[0138] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein R′ is hydrogen,methyl, ethyl, n-propyl, n-hexyl,

[0139] CO₂Et or (CH₂)₃OTBDPS. and X is a halogen. In certainembodiments, the invention provides the compound wherein R is acetyl andX is iodine.

[0140] The invention additionally provides a method of preparing anopen-chain aldehyde having the structure:

[0141] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R′ and R″ are independentlyhydrogen, a linear or branched alkyl, substituted or unsubstituted arylor benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, whichcomprises:

[0142] (a) cross-coupling a haloolefin having the structure:

[0143] wherein X is a halogen, with a terminal borane having thestructure:

[0144] wherein R*₂B is a linear, branched or cyclic alkyl or substitutedor unsubstituted aryl or benzyl boranyl moiety; and wherein Y is (OR₀)₂,(SR₀)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)— whereR₀ is a linear or branched alkyl, substituted or unsubstituted aryl orbenzyl; and wherein n is 2, 3 or 4, under suitable conditions to form across-coupled compound having the structure:

[0145] and

[0146] (b) deprotecting the cross-coupled compound formed in step (a)under suitable conditions to form the open-chain aldehyde. In certainembodiments, the invention provides the method wherein R is acetyl; R′is TBS; R″ is TPS; R*₂B is derived from 9-BBN; and Y is (OMe)₂.Cross-coupling step (a) is effected using reagents known in the artwhich are suited to the purpose. For example, the mixed borane may becross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂, orany known equivalent thereof, in the presence of such reagents as cesiumcarbonate and triphenylarsine. Deprotecting step (b) can be carried outusing a mild acid catalyst such as p-tosic acid, typically in a mixedaqueous organic solvent system, such as dioxane-water.

[0147] The invention also provides a method of preparing a protectedepothilone having the structure:

[0148] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkyl-arylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, which comprises:

[0149] (a) monoprotecting a cyclic diol having the structure:

[0150] under suitable conditions to form a cyclic alcohol havingstructure:

[0151] and

[0152] (b) oxidizing the cyclic alcohol formed in step (a) undersuitable conditions to form the protected epothilone. In certainembodiments, the invention provides the method wherein R′ and R″ areTBS. The monoprotecting step (a) may be effected using any of a varietyof suitable reagents, including TBSOTf in the presence of a base in aninert organic solvent. The base may be a non-nucleophilic base such as2,6-lutidine, and the solvent may be dichloromethane. The reaction isconducted at subambient temperatures, preferably in the range of −30° C.The oxidizing step (b) utilizes a selective oxidant such as Dess-Martinperiodinane in an inert organic solvent such as dichloromethane. Theoxidation is carried out at ambient temperatures, preferably at 20-25°C.

[0153] The invention further provides a method of preparing anepothilone having the structure:

[0154] which comprises:

[0155] (a) deprotecting a protected cyclic ketone having the structure:

[0156] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, under suitableconditions to form a desoxyepothilone having the structure:

[0157] and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone. In certain embodiments, theinvention provides the method wherein R′ and R″ are TBS. Deprotectingstep (a) is carried out by means of a treatment comprising a reagentsuch as HF•pyridine. The deprotected compound can be epoxidized in step(b) using an epoxidizing agent such acetic peracid, hydrogen peroxide,perbenzoic acid, m-chloroperbenzoic acid, but preferably withdimethyldioxirane, in an inert organic solvent such as dichloromethane.

[0158] The invention also provides a method of preparing a cyclic diolhaving the structure:

[0159] wherein R′ is a hydrogen, a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises:

[0160] (a) cyclizing an open-chain aldehyde having the structure:

[0161] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R″ is a hydrogen, a linearor branched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl under suitableconditions to form an enantiomeric mixture of a protected cyclic alcoholhaving the structure:

[0162] said mixture comprising an α- and a β-alcohol component;

[0163] (b) optionally isolating and oxidizing the α-alcohol formed instep (a) under suitable conditions to form a ketone and thereafterreducing the ketone under suitable conditions to form an enantiomericmixture of the protected cyclic alcohol comprising substantially theβ-alcohol; and

[0164] (c) treating the protected cyclic alcohol formed in step (a) or(b) with a deprotecting agent under suitable conditions to form thecyclic diol. In certain embodiments, the invention provides the methodwherein R′ is TBS and R″ is TPS. Cyclizing step (a) is performed usingany of a variety of mild nonnucleophilic bases such as KHMDS in an inertsolvent such as THF. The reaction is carried out at subambienttemperatures, preferably between −90° C. and −50° C., more preferably at−78° C. Isolation of the unnatural alpha-OH diastereomer is effected byany purification method, including any suitable type of chromatographyor by crystallization. Chromatographic techniques useful for the purposeinclude high pressure liquid chromatography, countercurrentchromatography or flash chromatography. Various column media are suited,including, inter alia, silica or reverse phase support. The beta-OHderivative is then oxidized using a selective oxidant, such asDess-Martin periodinane. The resulting ketone is the reduced using aselective reductant. Various hydridoborane and aluminum hydride reagentsare effective. A preferred reducing agent is sodium borohydride.Treating step (c ) may be effected using a variety of deprotectingagents, including HF-pyridine.

[0165] In addition, the invention provides a method of treating cancerin a subject suffering therefrom comprising administering to the subjecta therapeutically effective amount of any of the analogues related toepothilone B disclosed herein optionally in combination with apharmaceutically suitable carrier. The method may be applied where thecancer is a solid tumor or leukemia. In particular, the method isapplicable where the cancer is breast cancer or melanoma.

[0166] The subject invention also provides a pharmaceutical compositionfor treating cancer comprising any of the analogues of epothilonedisclosed hereinabove, as an active ingredient, optionally thoughtypically in combination with a pharmaceutically suitable carrier. Thepharmaceutical compositions of the present invention may furthercomprise other therapeutically active ingredients.

[0167] The subject invention further provides a method of treatingcancer in a subject suffering therefrom comprising administering to thesubject a therapeutically effective amount of any of the analogues ofepothilone disclosed hereinabove and a pharmaceutically suitablecarrier. The method is especially useful where the cancer is a solidtumor or leukemia.

[0168] The compounds taught above which are related to epothilones A andB are useful in the treatment of cancer, and particularly, in caseswhere multidrug resistance is present, both in vivo and in vitro. Theability of these compounds as non-substrates of MDR in cells, asdemonstrated in the Tables below, shows that the compounds are useful totreat, prevent or ameliorate cancer in subjects suffering therefrom.

[0169] The magnitude of the therapeutic dose of the compounds of theinvention will vary with the nature and severity of the condition to betreated and with the particular compound and its route ofadministration. In general, the daily dose range for anticancer activitylies in the range of 0.001 to 25 mg/kg of body weight in a mammal,preferably 0.001 to 10 mg/kg, and most preferably 0.001 to 1.0 mg/kg, insingle or multiple doses. In unusual cases, it may be necessary toadminister doses above 25 mg/kg.

[0170] Any suitable route of administration may be employed forproviding a mammal, especially a human, with an effective dosage of acompound disclosed herein. For example, oral, rectal, topical,parenteral, ocular, pulmonary, nasal, etc., routes may be employed.Dosage forms include tablets, troches, dispersions, suspensions,solutions, capsules, creams, ointments, aerosols, etc.

[0171] The compositions include compositions suitable for oral, rectal,topical (including transdermal devices, aerosols, creams, ointments,lotions and dusting powders), parenteral (including subcutaneous,intramuscular and intravenous), ocular (ophthalmic), pulmonary (nasal orbuccal inhalation) or nasal administration. Although the most suitableroute in any given case will depend largely on the nature and severityof the condition being treated and on the nature of the activeingredient. They may be conveniently presented in unit dosage form andprepared by any of the methods well known in the art of pharmacy.

[0172] In preparing oral dosage forms, any of the unusual pharmaceuticalmedia may be used, such as water, glycols, oils, alcohols, flavoringagents, preservatives, coloring agents, and the like in the case of oralliquid preparations (e.g., suspensions, elixers and solutions); orcarriers such as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, etc., inthe case of oral solid preparations are preferred over liquid oralpreparations such as powders, capsules and tablets. If desired, capsulesmay be coated by standard aqueous or non-aqueous techniques. In additionto the dosage forms described above, the compounds of the invention maybe administered by controlled release means and devices.

[0173] Pharmaceutical compositions of the present invention suitable fororal administration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient in powder or granular form or as a solution or suspension inan aqueous or nonaqueous liquid or in an oil-in-water or water-in-oilemulsion. Such compositions may be prepared by any of the methods knownin the art of pharmacy. In general compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers, finely divided solid carriers, or both and then, if necessary,shaping the product into the desired form. For example, a tablet may beprepared by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas powder or granule optionally mixed with a binder, lubricant, inertdiluent or surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine, a mixture of the powderedcompound moistened with an inert liquid diluent.

[0174] The present invention will be better understood from theExperimental Details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the invention as described in the claims whichfollow thereafter. It will be understood that the processes of thepresent invention for preparing epothilones A and B, analogues thereofand intermediates thereto encompass the use of various alternateprotecting groups known in the art. Those protecting groups used in thedisclosure including the Examples below are merely illustrative.

EXAMPLE 1

[0175] THP Glycidol; 13

[0176] A solution of (R)-(+)-glycidol 12 (20 g; 270 mmol) and freshlydistilled 3,4-dihydro-2H-pyran (68.1 g; 810 mmol) in CH₂Cl₂ (900 ml) wastreated with pyridinium p-toluenesulfonate (2.1 g; 8.36 mmol) at rt andthe resulting solution was stirred for 16 h. Approximately 50% of thesolvent was then removed in vacuo and the remaining solution was dilutedwith ether (1 L). The organic layer was then washed with two portions ofsaturated aqueous sodium bicarbonate (500 ml), dried (Na₂SO₄), filtered,and concentrated. Purification of the residue by flash chromatography(silica, 25→50% ether:hexanes) afforded THP glycidol 13 (31.2 g; 73%) asa colorless liquid: IR(film): 2941, 1122, 1034 cm⁻¹; ¹H NMR(CDCl₃,500MHz) d 4.66(t, J=3.5Hz, 1H), 4.64 (t, J=3.5Hz, 1H), 3.93 (dd, J=11.7,3.1 Hz, 1H), 3.86 (m, 2H), 3.73 (dd, J=11.8, 5.03 Hz, 1H), 3.67 (dd,J=11.83, 3.4 Hz, 1H), 3.51 (m, 2H), 3.40 (dd, J=11.7, 6.4, 1H), 3.18 (m,2H), 2.80 (m, 2H), 2.67 (dd, J=5.2, 2.7 Hz, 1H), 2.58 (dd, J=5.0, 2.7Hz,1H), 1.82 (m, 2H), 1.73 (m, 2H), 1.52 (m, 4H); ¹³C NMR (CDCl₃,125MHz) d 98.9, 98.8, 68.5, 67.3, 62.4, 62.2, 50.9, 50.6, 44.6, 44.5, 30.5,30.4, 25.4, 19.3, 19.2; [a]_(D)=+4.98 (c=2.15, CHCl₃).

EXAMPLE 2

[0177] Alcohol 13a

[0178] Trimethylsilylacetylene (32.3 g; 329 mmol) was added via syringeto THF (290 ml), and the resulting solution was cooled to 78° C. andtreated with n-butyllithium (154 ml of a 1.6 M solution in hexanes;246.4 mmol). After 15 min, boron trifluoride diethyl etherate (34.9 g;246 mmol) was added, and the resulting mixture was stirred for 10 min. Asolution of epoxide 13 (26 g; 164.3 mmol) in THF (130 ml) was then addedvia a cannula and the resulting solution was stirred for 5.5 h at −78°C. The reaction was quenched by the addition of saturated aqueous sodiumbicarbonate solution (250 ml) and the solution was allowed to warm tort. The mixture was then diluted with ether (600 ml) and washedsuccessively with saturated aqueous sodium bicarbonate solution (250ml), water (250 ml), and brine (250 ml). The organic layer was thendried (Na₂SO₄), filtered, and concentrated in vacuo. Purification of theresidue by flash chromatography (silica, 20% ether:hexanes) providedalcohol 13a (34 g; 76%).

EXAMPLE 3

[0179] MOM Ether 13b

[0180] A solution of alcohol 13a (24 g; 88.9 mmol) andN,N-diisopropylethylamine (108 ml; 622 mmol) in anhydrous1,2-dichloroethane (600 ml) was treated with chloromethyl methyl ether(17 ml; 196 mmol), and the resulting mixture was heated to 55° C. for 28h. The dark mixture was then cooled to rt and treated with saturatedaqueous sodium bicarbonate solution (300 ml). The layers were separated,and the organic layer was washed successively with saturated aqueoussodium bicarbonate solution (200 ml) and brine (200 ml). The organiclayer was then dried (MgSO₄) and filtered through a pad of silica gel(ether rinse). Purification of the residue by flash chromatography(silica, 20→30% ether:hexanes) afforded MOM ether 13b (23.7 g; 85%) as apale yellow oil.

EXAMPLE 4

[0181] Alcohol 14

[0182] A solution of THP ether 13b (20 g; 63.7 mmol) in methanol (90 ml)was treated with pyridinium p-toluenesulfonate (4.0 g; 15.9 mmol) andthe resulting mixture was stirred at rt for 16 h. The reaction was thenquenched by the addition of saturated aqueous sodium bicarbonatesolution (100 ml), and the excess methanol was removed in vacuo. Theresidue was diluted with ether (300 ml), and the organic layer waswashed successively with saturated aqueous sodium bicarbonate solution(200 ml) and brine (200 ml). The organic layer was dried (MgSO₄),filtered, and concentrated. Purification of the residue by flashchromatography (silica, 40→50% ether:hexanes) provided alcohol 14 (13.1g; 95%) as a colorless oil.

EXAMPLE 5

[0183] Alcohol 14a

[0184] To a cooled (−78° C.) solution of oxalyl chloride (24.04 ml of a2.0 M solution in CH₂Cl₂; 48.08 mmol) in CH₂Cl₂ (165 ml) was addedanhydrous DMSO (4.6 ml; 64.1 mmol) in dropwise fashion. After 30 min, asolution of alcohol 14 (6.93 g; 32.05 mmol) in CH₂Cl₂ (65 ml+10 mlrinse) was added and the resulting solution was stirred at −78° C. for40 min. Freshly distilled triethylamine (13.4 ml; 96.15 mmol) was thenadded, the cooling bath was removed, and the mixture was allowed to warmto 0° C. The reaction mixture was then diluted with ether (500 ml), andwashed successively with two portions of water (250 ml) and one portionof brine (250 ml). The organic layer was then dried (MgSO₄), filtered,and concentrated.

[0185] The crude aldehyde (6.9 g) prepared in the above reaction wasdissolved in ether (160 ml) and cooled to 0° C. Methylmagnesium bromide(32.1 ml of a 3.0 M solution in butyl ether; 96.15 mmol) was then added,and the solution was allowed to warm slowly to rt. After 10 h, thereaction mixture was cooled to 0° C. and the reaction was quenched bythe addition of saturated aqueous ammonium chloride solution. Themixture was diluted with ether (200 ml) and washed successively withwater (150 ml) and brine (150 ml). The organic layer was dried (MgSO₄),filtered, and concentrated. Purification of the residue by flashchromatography (silica, 40→50% ether:hexanes) provided alcohol 14a (6.3g; 85% from 14).

EXAMPLE 6

[0186] Ketone 15

[0187] A solution of alcohol 14 (1.0 g; 4.35 mmol), 4 A mol. sieves, andN-methylmorpholine-N-oxide (1.0 g; 8.7 mmol) in CH₂Cl₂ (20 ml) at rt wastreated with a catalytic amount of tetra-n-propylammonium perruthenate,and the resulting black suspension was stirred for 3 h. The reactionmixture was then filtered through a pad of silica gel (ether rinse), andthe filtrate was concentrated in vacuo. Purification of the residue byflash chromatography (silica, 10% ether:hexanes) afforded ketone 15 (924mg; 93%) as a light yellow oil.

EXAMPLE 7

[0188] Alkene 17

[0189] A cooled (−78° C.) solution of phosphine oxide 16 (1.53 g; 4.88mmol) in THF (15.2 ml) was treated with n-butyllithium (1.79 ml of a2.45 M solution in hexanes). After 15 min, the orange solution wastreated with a solution of ketone 15 (557 mg; 2.44 mmol) in THF (4.6ml). After 10 min, the cooling bath was removed, and the solution wasallowed to warm to rt. The formation of a precipitate was observed asthe solution warmed. The reaction was quenched by the addition ofsaturated aqueous ammonium chloride solution (20 ml). The mixture wasthen poured into ether (150 ml) and washed successively with water (50ml) and brine (50 ml). The organic layer was dried (Na₂SO₄), filtered,and concentrated.

[0190] Purification of the residue by flash chromatography (silica, 10%ether:hexanes) afforded alkene 17 (767 mg; 97%) as a colorless oil:IR(film): 2956, 2177, 1506, 1249, 1149, 1032, 842, cm⁻¹; ¹H NMR(CDCl₃,500MHz) d 6.95(s, 1H), 6.53(s, 1H), 4.67(d, J=6.7Hz, 1H), 4.57 (d, J=6.8Hz, 1H), 4.29 (dd, J=8.1, 5.4 Hz, 1H), 3.43 (s, 3H), 2.70 (s, 3H), 2.62(dd, J=16.9, 8.2 Hz, 1H), 2.51(dd, J=17.0,5.4 Hz, 1H), 2.02(s, 3H); ¹³CNMR (CDCl₃, 125 MHz) d 164.4, 152.5, 137.1, 121.8, 116.2, 103.7, 93.6,86.1, 79.6, 55.4, 25.9, 19.1, 13.5; [α]_(D)=−27.3 (c=2.2, CHCl₃).

EXAMPLE 8

[0191] Alkynyl iodide formation

[0192] To a solution of the alkyne 17 (3.00 g, 9.29 mmol) in acetone(100 mL) at 0° C. was added NIS (2.51 g; 11.2 mmol) and AgNO₃ (0.160 g;0.929 mmol). The mixture was then slowly warmed to rt. After 1.5 h, thereaction was poured into Et₂O (250 mL) and washed once with satbisulfite (40 mL), once with sat NaHCO₃ (40 mL), once with brine (40 mL)and dried over anhydrous MgSO₄. Purification by flash chromatography onsilica gel using gradient elution with hexanes/ethyl acetate (10:1-7:1)gave 2.22 g (64%) of the iodide 17a as an amber oil.

EXAMPLE 9

[0193] Reduction of the alkynyl iodide:

[0194] BH₃•DMS (0.846 mL, 8.92 mmol) was added to a solution ofcyclohexene (1.47 mL, 17.9 mmol) in Et₂O (60 mL) at 0° C. The reactionwas then warmed to rt. After 1 h, the iodide x (2.22 g, 5.95 mmol) wasadded to Et₂O. After 3 h, AcOH (1.0 mL) was added. After 30 additionalmin, the solution was poured into sat NaHCO₃ and extracted with Et₂O(3×100 mL). The combined organics were then washed once with brine (50mL) and dried over anhydrous MgSO₄. Purification by flash chromatographyon silica gel eluting with hexanes/ethyl acetate (6:1) gave 1.45 g (65%)of the vinyl iodide 18 as a yellow oil.

EXAMPLE 10

[0195] MOM removal:

[0196] To a solution of iodide 18 (1.45 g, 3.86 mmol) in CH₂Cl₂ (40 mL)at rt was added thiophenol (1.98 mL, 19.3 mmol) and BF₃∘Et₂O (1.90 mL,15.43 mmol). After 22 h, the reaction was poured into EtOAc (150 mL) andwashed with 1 N NaOH (2×50 mL) and dried over anhydrous MgSO₄.Purification by flash chromatography on silica gel using gradientelution with hexanes/ethyl acetate (4:1-2:1-1:1) gave 1.075 g (86%) ofthe alcohol 18a as a pale yellow oil.

EXAMPLE 11

[0197] Acetate Formation

[0198] To a solution of alcohol 18a (1.04 g, 3.15 mmol) in CH₂Cl₂ (30mL) was added pyridine (2.52 mL, 25.4 mmol), acetic anhydride (1.19 mL,12.61 mmol) and DMAP (0.005 g). After 1 h, the volatiles were removed invacuo. Purification of the resulting residue by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (7:1 ) gave 1.16 g (99%)of the acetate 19 as a pale yellow oil. IR(film):1737, 1368, 1232, 1018cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 6.97 (s, 1H), 6.53 (s,1H), 6.34 (dd,J=17.5, 1.0 Hz, 1H), 6.18 (dd, J=13.7, 6.9 Hz, 1h), 5.40 (t, J=6.4 Hz,1H), 2.70 (s, 3h), 2.61 (m, 2H), 2.08 (2s, 6H). ¹³C NMR (CDCl₃,125 MHz)d 169.8, 164.4, 152.2, 136.4, 136.1, 120.6, 116.4, 85.1, 38.3, 21.0,19.1, 14.7; [α]_(D)=−28.8 (c=1.47, CHCl₃).

EXAMPLE 12

[0199] To a solution of alcohol 4 (2.34 g, 3.62 mmol) and 2,6-lutidine(1.26 mL, 10.86 mmol) in CH₂Cl₂ (23 mL) at 0° C. was treated with TBSOTf(1.0 mL, 4.34 mmol). After stirrring for 1.5 at 0° C. the reactionmixture was quenched with MeOH (200 uL) and the mixture stirred anadditional 5 min. The reaction mixture was diluted with Et₂O (100 mL)and washed successively with 1 N HCl (25 mL), water (25 mL), and brine(25 mL). The solution was dried over MgSO₄, filtered, and concentrated.The residue was purified by flash chromatography on silica gel elutingwith 5% Et₂O in hexanes to provide compund 7 (2.70 g, 98%) as acolorless foam.

EXAMPLE 13

[0200] A solution of compound 7 (2.93 g, 3.85 mmol) in CH₂Cl₂/H₂O (20:1,80 mL) was treated with DDQ (5.23 g, 23.07 mmol) and the resultingsuspension was stirred at room temperature for 24 h. The reactionmixture was diluted with Et₂O (200 mL) and washed with aqueous NaHCO₃(2×40 mL). The aqueous layer was extracted with Et₂O (3×40 mL) and thecombined organic fractions were washed with brine (50 mL), dried overMgSO₄, filtered, and concentrated. Purification of the crude oil byflash chromatography on silica gel eluting with 30% ether in hexanesafforded alcohol 7A (2.30 g, 89%) as a colorless oil: IR (film) 3488,1471, 1428, 1115, 1054 cm⁻; ¹H NMR (CDCl₃, 500 MHz) d 7.70 (6H, dd,J=8.0, 1.5 Hz), 7.44 (9H, s), 4.57 (1H, d, J=3.5 Hz), 4.19 (1H, s), 3.67(1H, d, J=8.5 Hz), 3.06 (1H, dd, J=11.5, 5.0 Hz), 2.89 (1H, dd, J=11.5,5.0 Hz), 2.68 (1H, d, J=13.5 Hz), 2.59 (1H, d, J=13.5 Hz), 2.34 (1H, dt,J=12.0, 2.5 Hz), 2.11 (1H, m), 1.84 (1H, dt, J=12.0, 2.5 Hz), 1.76 (2H,m), 1.59 (2H, m), 1.34 (3H, s), 1.13 (3H, d, J=7.5 Hz), 1.10 (3H, s),0.87 (9H, s), 0.84 (3H, d, J=12.0 Hz), 0.02 (3H, s), 0.01 (3H, s); ¹³CNMR (CDCl₃, 125 MHz) d 136.18, 134.66, 130.16, 127.84, 78.41, 75.91,63.65, 59.69, 45.43, 45.09, 37.72, 30.84, 30.50, 26.23, 25.89, 22.42,21.05, 18.40, 15.60, 14.41, −3.23, −3.51; [α]_(D)=−0.95 (c=0.173,CHCl₃).

EXAMPLE 14

[0201] To a solution of oxalyl chloride (414 μL, 4.74 mmol) in CH₂Cl₂(40 mL) at −78° C. was added dropwise DMSO (448 uL, 6.32 mmol) and theresulting solution was stirred at −78° C. for 30 min. Alcohol 7a (2.12g, 3.16 mmol) in CH₂Cl₂ (20 mL) was added and the resulting whitesuspension was stirred at −78° C. for 45 min. The reaction mixture wasquenched with Et₃N (2.2 mL, 15.8 mmol) and the solution was allowed towarm to 0° C. and stirred at this temperature for 30 min. The reactionmixture was diluted with Et₂O (100 mL) and washed successively withaqueous NH₄Cl (20 mL), water (20 mL), and brine (20 mL). The crudealdehyde was purified by flash chromatography on silica gel eluting with5% Et₂O in hexanes to provide aldehyde 8 (1.90 g, 90%) as a colorlessoil.

EXAMPLE 15

[0202] A solution of (methoxymethyl)triphenylphosphonium chloride (2.97g, 8.55 mmol) in THF (25 mL) at 0° C. was treated with KO^(t)Bu (8.21mL, 1M in THF, 8.1 mmol). The mixture was stirred at 0° C. for 30 min.Aldehyde 8 (3.1 g, 4.07 mmol) in THF (10 mL) was added and the resultingsolution was allowed to warm to room temperature and stirred at thistemperature for 2 h. The reaction mixture was quenched with aqueousNH₄Cl (40 mL) and the resulting solution extracted with Et₂O (3×30 mL).The combined Et₂O fractions were washed with brine (20 ml), dried overMgSO₄, filtered, and concentrated. The residue was purified by flashchromatography on silica gel eluting with 5% Et₂O in hexanes to yieldcompound 9 (2.83 g, 86%) as a colorless foam.

EXAMPLE 16

[0203] To a solution of compound 9 (2.83g, 3.50 mmol) in dioxane/H₂O(9:1, 28 mL) was added pTSA•H₂O (1.0 g, 5.30 mmol) and the resultingmixture was heated to 50° C. for 2 h. After cooling to room temperaturethe mixture was diluted with Et₂O (50 mL) and washed with aqueous NaHCO₃(15 mL), brine (20 ml), dried over MgSO₄, filtered, and concentrated toprovide aldehyde 9a (2.75 g, 99%) as a colorless foam.

EXAMPLE 17

[0204] Methyltriphenylphosphonium bromide (1.98 g, 5.54 mmol) in THF (50mL) at 0° C. was treated with lithium bis(trimethylsilyl)amide (5.04 mL,1M in THF, 5.04 mmol) and the resulting solution was stirred at 0° C.for 30 min. Aldehyde 9a (2.0 g, 2.52 mmol) in THF (5.0 mL) was added andthe mixture was allowed to warm to room temperature and stirred at thistemperature for 1 h. The reaction mixture was quenched with aqueousNH₄Cl (15 mL) and extracted with Et₂O (3×20 mL). The combined Et₂Ofractions were washed with brine (15 mL), dried over MgSO₄, filtered,and concentrated. The residue was purified by flash chromatography onsilica gel eluting with 5% Et₂O in hexanes to afford compound 10 (1.42g, 76%) as a colorless foam.

EXAMPLE 18

[0205] A solution of compound 10 (1.0 g, 1.34 mmol) in MeOH/THF (2:1, 13mL) was treated with [bis(trifluoroacetoxy)iodobenzene] (865 mg, 2.01mmol) at room temperature. After 15 min the reaction mixture wasquenched with aqueous NaHCO₃ (25 mL). The mixture was extracted withEt₂O (3×25 mL) and the combined Et₂O fractions were washed with brine,dried over MgSO₄, filtered, and concentrated. Purification of theresidue by flash chromatography on silica gel eluting with 5% Et₂O inhexanes provided compound 11 (865 mg, 92%) as a colorless foam: IR(film) 1428, 1252, 1114, 1075, 1046 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 7.61(6H, dd, J=7.9, 1.4 Hz), 7.38 (9H, s), 5.47 (1H, m), 4.87 (1H, d, J=10.0Hz), 4.76 (1H, d, J=15.9 Hz), 4.30 (1H, d, J=3.7 Hz), 3.95 (1H, s), 3.56(1H, dd, J=7.5, 1.4 Hz), 3.39 (3H, s), 2.84 (3H, s), 2.02 (1H, m), 1.64(2H, m), 1.34 (1H, m), 1.11 (3H, s), 1.02 (3H, d, J=7.4 Hz), 0.90 (3H,s), 0.85 (9H, s), 0.62 (3H, d, J=6.8 Hz), −0.04(3H, s), −0.05 (3H, s);¹³C NMR (CDCl₃, 125 MHz) d 138.29, 135.79, 135.04, 129.86, 127.78,114.98, 110.49, 60.11, 55.57, 46.47, 43.91, 36.82, 34.21, 26.26, 19.60,18.60, 17.08, 16.16, 13.92, −2.96, −3.84; [α]_(D)=+1.74 (c=0.77, CHCl₃).

EXAMPLE 19

[0206] Suzuki Coupling

[0207] To a solution of olefin 11 (0.680 g, 1.07 mmol) in THF (8.0 mL)was added 9-BBN (0.5 M soln in THF, 2.99 mL, 1.50 mmol). In a separateflask, the iodide 19 (0.478 g, 1.284 mmol) was dissolved in DMF (10.0mL). CsCO₃ (0.696 g, 2.14 mmol) was then added with vigorous stirringfollowed by sequential addition of Ph₃As (0.034 g, 0.111 mmol),PdCl₂(dppf)₂ (0.091 g, 0.111 mmol) and H₂O (0.693 mL, 38.5 mmol). After4 h, then solution was added to the iodide mixture in DMF. The reactionquickly turned dark brown in color and slowly became pale yellow after 2h. The reaction was then poured into H₂O (100 mL) and extracted withEt₂O (3×50 mL). The combined organics were washed with H₂O (2×50 mL),once with brine (50 mL) and dried over anhydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(7:1) gave 0.630 g (75%) of the coupled product 20 as a pale yellow oil.

EXAMPLE 20

[0208] Hydrolysis of Dimethyl Acetal 21

[0209] The acetate 20 (0.610 g, 0.770 mmol) was dissolved in dioxane/H₂O(9:1, 15 mL) and p-TSA.H₂O (0.442 g, 2.32 mmol) was added. The mixturewas then heated to 55° C. After 3 h, the mixture was cooled to rt andpoured into Et₂O. This solution was washed once with sat NaHCO₃ (30 mL),once with brine (30 mL) and dried over anhydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(7:1) gave 0.486 g (85%) of the aldehyde 21 as a pale yellow oil. IR(film) 1737, 1429, 1237, 1115, 1053 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) d9.74(1H, s), 7.61 (6H, dd, J=7.8, 1.2 Hz), 7.38 (9H, m), 6.94 (1H, s),6.53 (1H, s), 5.39 (1H, m), 5.29 (1H, t, J=6.9 Hz), 4.61 (1H, d, J=4.3Hz), 3.50 (1H, dd, J=5.2, 2.6 Hz), 2.70 (3H, s), 2.48 (2H, m), 2.14 (1H,m), 2.09 (3H, s), 2.07 (3H, s), 1.83 (2H, m), 1.41 (1H, m), 1.18 (1H,m), 1.01 (3H, s), 0.99 (3H, s), 0.91 (3H, d, J=7.4 Hz), 0.85 (9H, s),0.69 (1H, m), 0.58 (3H, d, J=6.8 Hz), −0.05 (3H, s), −0.06 (3H, s); ¹³CNMR (CDCl₃, 125 MHz) d 205.46, 170.01, 164.49, 152.46, 137.10, 135.60,134.22, 132.55, 130.65, 127.84, 123.82, 120.66, 116.19, 81.09, 78.47,76.73, 51.66, 43.14, 38.98, 30.99, 30.42, 27.63, 26.10, 21.15, 20.92,20.05, 19.15, 18.49, 15.12, 14.70, 12.75, −3.25, −4.08; [α]_(D)=−18.7(c=0.53, CHCl₃).

EXAMPLE 21

[0210] Aldol

[0211] To a solution of the acetate-aldehyde 21 (84 mg,0.099 mmol) inTHF at −78° C. was added KHMDS (0.5M in toluene, 1.0 ml, 0.5 mmol))dropwise.The resulting solution was stirred at −78° C. for 30 min. Thenthe reaction mixure was cannulated to a short pad of silica gel andwashed with ether. The residue was purified by flash chromatography(silica, 12% EtOAc in hexane) to give the lactone 22 (37 mg of 3-S and 6mg of 3-R, 51%) as white foam.

EXAMPLE 22

[0212] Monodeprotection

[0213] Lactone 22 (32 mg, 0.0376 mmol) was treated with 1 ml of pyridinebuffered HF•pyridine—THF solution at room temperture for 2 h. Thereaction mixure was poured into saturated aqueous NaHCO₃ and extractedwith ether . The organic layer was washed in sequence with saturatedCuSO₄ (10 ml×3) and saturated NaHCO₃ (10 ml), then dried over Na₂SO₄ andconcentrated under vacuum. The residue was purified by flashchromatography (silica, 25% EtOAc in hexane) and to give diol 22a (22mg, 99%) as white foam.

EXAMPLE 23

[0214] TBS-Protection

[0215] To a cooled (−30° C.) solution of diol 22a (29 mg, 0.0489 mmol)and 2,6-lutidine (0.017 ml, 0.147 mmol) in anhydrous CH₂Cl₂(1 ml) wasadded TBSOTf (0.015 ml, 0.0646 mmol). The resulting solution was thenstirred at −30° C. for 30 min. The reaction was quenched with 0.5M HCl(10 ml) and extracted with ether (15 ml). Ether layer was washed withsaturated NaHCO₃, dried (Na₂SO₄) and concentrated in vacuo. Purifictionof the residue by flash chromatogrphy (silica, 8% EtOAc in hexane)afforded TBS ether 22B (32 mg, 93%) as white foam.

EXAMPLE 24

[0216] Ketone Formation

[0217] To a solution of alcohol 22B (30 mg, 0.0424 mmol) in CH₂Cl₂ (2.0mL) at 25° C. was added Dess-Martin periodinane (36 mg, 0.0848 mmol) inone portion. The resulting solution was then allowed to stir at 25° C.for 1.5 h. The reaction was quenched by the addition of 1:1 saturatedaqueous sodium bicarbonate:sodium thiosulfate (10 ml) and stirred for 5min. The mixture was then extracted with ether (3×15 ml). The organiclayer was dried (Na₂SO₄), filtered, and concentrated in vacuo .Purification of the residue by flash chromatography (silica, 8% EtOAc inhexane) provided ketone 22C (25 mg, 84%) as white foam. IR(film): 2928,1745, 1692, 1254, 1175, 836 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) d 6.97 (s, 1H),6.57 (s, 1H), 5.53 (dt, J=3.4, 11.1 Hz, 1H), 5.37 (dd, J=16.4, 9.9 Hz,1H), 5.00 (d, J=10.3 Hz, 1H), 4.02 (d, J=9.7 Hz, 1H), 3.89 (d, J=8.7 Hz,1H), 3.00 (m, 1H), 2.82 (d, J=6.5 Hz, 1H), 2.71 (m, 5H), 2.36 (q, J=10.7Hz, 1H), 2.12 (, 3H), 2.07 (dd, J−8.2, 1H), 1.87 (bs, 1H), 1.49 (m, 3H),1.19 (m, 5H), 1.14 (s, 3H), 1.08 (d, J=6.8 Hz, 3H), 0.94 (m, 12H), 0.84(s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.07 (s, 3H), −0.098 (s, 3H); ¹³CNMR (CDCl₃, 125 MHz) d 218.7, 170.1, 164.5, 152.6, 137.9, 133.9, 124.8,119.6, 115.9, 72.7, 53.2, 43.9, 41.0, 40.3, 32.9, 32.3, 28.4, 27.1,26.3, 26.1, 26.0, 19.2, 19.1, 18.3, 18.2, 17.1, 16.0, 15.2, 14.3, −4.2,−4.4, −4.6, −4.8 ; [α]_(D)=−21.93 (c=1.4, CHCl₃).

EXAMPLE 25

[0218] Desoxy Compound

[0219] To a solution of TBS ether 22C (27 mg, 0.038 mmol) in THF(1 ml)at 25° C. in a plastic vial was added dropwise HF•pyridine (0.5 ml). Theresulting solution was allowed to stir at 25° C. for 2 h. The reactionmixture was diluted with chloroform (2 ml) and very slowly added tosatured sodium bicarbonate (20 ml) . The mixture was extracted withCHCl₃ (20ml×3). The organic layer was dried (Na ₂SO₄), filtered, andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 30% EtOAc in hexane) provided diol 23 (18 mg,99%) as white foam: IR(film): 3493, 2925, 1728, 1689, 1249 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) d 6.96 (s, 1H), 6.59 (s, 1H), 5.44 (dt, J=4.3, 10.4 Hz,1H), 5.36 (dt, J=5.1, 10.2 Hz, 1H), 5.28 (dd, J=1.7, 9.8 Hz, 1H), 4.11(d, J=7.2 Hz, 1H), 3.74 (s, 1H), 3.20 (d, J=4.5 Hz, 1H), 3.14 (dd,J=2.2, 6.8 Hz, 1H), 3.00 (s, 1H), 2.69 (m, 4H), 2.49 (dd, J=11.3, 15.1Hz, 1H), 2.35 (dd, J−2.5, 15.1 Hz, 1H), 2.27 (m, 1H), 2.05 (m, 1H), 2.04(s, 3H), 2.01 (m, 1H) 1.75 (m, 1H), 1.67 (m, 1H), 1.33 (m, 4H), 1.21 (s,1H), 1.19 (m, 2H), 1.08 (d, J=7.0 Hz, 3H), 1.00 (s, 3H), 0.93 (d, J=7.1Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz) d 226.5 176.5, 171.1, 158.2, 144.7,139.6, 131.1, 125.7, 122.0, 84.6, 80.2, 78.6, 59.4, 47.9, 45.4, 44.6,38.5, 37.9, 33.7, 33.6, 28.7, 25.1, 25.0, 21.9, 21.7, 19.6;[α]_(D)=−84.7 (c=0.85, CHCl₃).

EXAMPLE 26

[0220] Epothilone

[0221] To a cooled (−50° C.) solution of desoxyepothilone (9 mg, 0.0189mmol) in dry CH₂Cl₂ (1 ml) was added freshly prepared dimethyldioxirane(0.95 ml, 0.1 M in acetone). The resulting solution was allowed to warmup to −30° C. for 2 h. A stream of nitrogen was then bubbled through thesolution to remove excess DMDO. The residue was purified by flashchromatography (silica, 40% EtOAc in hexane) and afforded epothilone A(4.6 mg, 49%) as colorless solid and 0.1 mg of cis-epoxide diastereomer.This material was identical with the natural epothilone A in allrespects.

EXAMPLE 27

[0222] Procedure for Ring-closing Olefin Metathesis

[0223] To a stirred solution of diene 24 (5 mg, 0.0068 mmol) in drybenzene (1.5 mL) was added Grubbs's catalyst (2.8 mg, 0.0034 mmol).After 12 h, an additional portion of catalyst was added (2.8 mg). Afteran additional 5 h, the reaction was concentrated. Purification by silicagel chromatography eluting with hexanes/ethyl acetate (11:1) gave thelactone 23 (3.5 mg, 94%, 2:1 E/Z).

EXAMPLE 28

[0224] Preparation of Compound 19

[0225] Alcohol 2A

[0226] A mixture of (S)-(−)-1,1,¹-bi-2-naphthol (259 mg. 0.91 mmol),Ti(O-i-Pr)₄ (261 μL; 0.90 mmol), and 4 Å sieves (3.23 g) in CH₂Cl₂ (16mL) was heated at reflux for 1 h. The mixture was cooled to rt andaldehyde 1 was added. After 10 min. the suspension was cooled to −78°C., and allyl tributyltin (3.6 mL; 11.60 mmol) was added. The reactionmixture was stirred for 10 min at −78° C. and then placed in a −20° C.freezer for 70 h. Saturated NaHCO₃ (2 mL) was added, and the mixture wasstirred for 1 h, poured over Na₂SO₄, and then filtered through a pad ofMgSO₄ and celite. The crude material was purified by flashchromatography (hexanes/ethyl acetate, 1:1) to give alcohol 2A as ayellow oil (1.11g; 60%).

EXAMPLE 29

[0227] Acetate 3A

[0228] To a solution of alcohol 2A (264 mg; 1.26 mmol) in CH₂Cl₂ (12 mL)was added DMAP (15 mg: 0.098 mmol), Et₃N (0.45 mL; 3.22 mmol), and Ac₂O(0.18 mL; 1.90 mmol). After 2 h, the reaction mixture was quenched by 20mL of H₂O, and extracted with EtOAC (4×20 mL). The combined organiclayer was dried with MgSO₄, filtered, and concentrated. Flashchromatrography (EtOAC/hexanes, 1:3) afforded acetate 3A as a yellow oil(302 mg; 96%).

EXAMPLE 30

[0229] Vinyl Iodide 19

[0230] To a solution of acetate 3A (99 mg; 0.39 mmol) in acetone at 0°C. was added H₂O (4 drops), OsO₄ (2.5% wt. in butyl alcohol; 175 μL;0.018 mmol), and N-methyl-morpholine-N-oxide (69 mg; 0.59 mmol). Themixture was stirred at 0° C. for 2 h and 45 min and then quenched withNa₂SO₃. The solution was poured to 10 mL of H₂O and extracted with EtOAc(5×10 mL). The combined organic layer was dried over MgSO4, filtered,and concentrated.

[0231] To a solution of this crude product in THF/H₂O (4 mL, 3:1) wasadded NalO₄ (260 mg; 1.22 mmol). After 1.25 h, the reaction mixture wasthen quenched with 10 mL of H₂O and concentrated. The residue wasextracted with EtOAc (5×10 mL). The organic layer was dried over MgSO₄,filtered, and concentrated. Flash chromatography (EtOAc/hexanes, 1:1)gave a yellow oil (80 mg) which contained unidentified by-product(s).This mixture was used without further purification.

[0232] To a solution of (Ph₃P ⁺CH₂I)I⁻ (100 mg; 0.19 mmol) in 0.25 mL ofTHF at rt was added 0.15 mL (0.15 mmol) of NaHMDS (1M in THF). To theresulting solution at −78° C. was added HMPA (22 μL; 0.13 mmol) and theproduct from previous step (16 mg) in THF (0.25 mL). The reactionmixture was then stirred at rt for 30 min. After the addition of hexanes(10 mL), the solution was extracted with EtOAc (4×10 mL). The combinedEtOAC layer was dried (MgSO₄), filtered, and concentrated. PreparativeTLC (EtOAc/hexanes, 2.3) afforded vinyl iodide 19 as a yellow oil (14mg; 50% for three steps).

EXAMPLE 31

[0233] Iodoolefin Acetate 8C

[0234] To a suspension of ethyltriphenylphosphonium iodide (1.125 g,2.69 mmol) in THF (10 mL) was added nBuLi (2.5 M soln in hexanes, 1.05mL, 2.62 mmol) at rt. After disappearance of the solid material, thesolution was added to a mixture of iodine (0.613 g, 2.41 mmol) in THF(20 mL) at −78° C. The resulting suspension was vigorously stirred for 5min at −78° C., then warmed up −20° C., and treated with sodiumhexamethyldisilazane (1 M soln in THF, 2.4 mL, 2.4 mmol). The resultingred solution was stirred for 5 min followed by the slow addition ofaldehyde 9C (0.339 g, 1.34 mmol). The mixture was stirred at −20° C. for40 min, diluted with pentane (50 mL), filtered through a pad of celite,and concentrated. Purification of the residue by flash columnchromatography (hexanes/ethyl acetate, 85:15) gave 0.202 g (25% overallfrom vinyl acetate 10C) of the vinyl iodide 8C as a yellow oil. IR(film): 2920, 1738, 1234 cm⁻¹; ¹H NMR (CDCl₃): δ 6.98 (s, 1H), 6.56 (s,1H), (dd, J=5.43, 6.57 Hz, 1H), 5.35 (t, J=6.6 Hz, 1H), 2.71(s, 3H),2.54 (q, J=6.33, 2H), 2.50 (s, 3H), 2.09 (s, 6H); ¹³C NMR (CDCl₃): δ170.1, 164.6, 152.4, 136.9, 130.2, 120.6, 116.4, 103.6, 40.3, 33.7,21.2, 19.2, 14.9; [α]_(D)=−20.7° (c=2.45, CHCl₃).

EXAMPLE 32

[0235] Acetal 13C

[0236] To a solution of olefin “7C” (0.082 g, 0.13 mmol) in THF (0.5 mL)was added 9-BBN (0.5 M soln in THF, 0.4 mL, 0.2 mmol). After stirring atrt. for 3.5 h, an additional portion of 9-BBN (0.5 M soln in THF, 0.26mL, 0.13 mmol) was added. In a separate flask, iodide 8C (0.063 g, 0.16mmol) was dissolved in DMF (0.5 mL). Cs₂CO₃ (0.097 g, 0.30 mmol) wasthen added with vigorous stirring followed by sequential addition ofPdCl₂(dppf)₂ (0.018 g, 0.022 mmol), Ph₃As (0.0059 g, 0.019 mmol), andH₂O (0.035 mL, 1.94 mmol). After 6 h, then borane solution was added tothe iodide mixture in DMF. The reaction quickly turned dark brown incolor and slowly became pale yellow after 3 h. The reaction was thenpoured into H₂O (10 mL) and extracted with Et₂O (3×15 mL). The combinedorganic layers were washed with H₂O (3×15 mL), brine (1×20 mL), driedover MgSO₄, filtered, and concentrated. Flash column chromatography(hexanes/ethyl acetate, 9:1) gave 0.089 g (77%) of the coupled product13C as a yellow oil.

EXAMPLE 33

[0237] Aldehyde 14C

[0238] Acetal 13C (0.069 g, 0.077 mmol) was dissolved in dioxane/H₂O(9:1, 1 mL) and pTSA•H₂O (0.045 g, 0.237 mmol) was added. The mixturewas then heated to 55° C. After 3 h, the mixture was cooled to rt,poured into Et₂O, and extracted with Et₂O (4×15 mL). The combined ethersolutions were washed with sat NaHCO₃ (1×30 mL), brine (1×30 mL), driedover MgSO₄, filtered, and concentrated. Flash column chromatography(hexanes/ethyl acetate, 3:1) gave 0.046 g (71%) of the aldehyde 14C as apale yellow oil.

EXAMPLE 34

[0239] Macrocycle 15C-(SR)

[0240] To a solution of aldehyde 14C (0.021 g, 0.024 mmol) in THF (5 mL)at −78° C. was added KHMDS (0.5 M soln in toluene, 0.145 mL, 0.073mmol). The solution was stirred at −78° C. for 1 h, then quenched withsat'd NH₄Cl, and extracted with ether (3×15 mL). The combined organiclayers were dried with MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 7:1) gave 0.008 g of the desiredα-alcohol 15C-(S) and 0.006 g of β-alcohol 15C-(R) (67% total ) as paleyellow oils.

EXAMPLE 35

[0241] Macrocycle 15C-(S)

[0242] To a solution of β-alcohol 15C-(R) (0.006 g, 0.0070 mmol) in 0.5mL of CH₂Cl₂ at rt. was added Dess-Martin periodinane (0.028g, 0.066mmol). After 0.5 h, an additional portion of Dess-Martin periodinane(0.025 mg, 0.059 mmol) was added. The resulting solution was stirred atrt for additional 1 h, then treated with ether (2 mL) and sat'dNa₂S₂O₃/sat'd NaHCO₃ (3 mL, 1:1), poured into H₂O (20 mL), and extractedwith ether (4×10 mL). The combined ether solutions were washed with H₂O(1×30 mL), brine (1×30 mL), dried with MgSO₄, filtered, andconcentrated. To a solution of crude ketone 15C′ in MeOH/THF (2 mL, 1:1)at −78° C. was added NaBH₄ (0.015 g, 0.395 mmol). The resulting solutionwas stirred at rt for 1 h, quenched with sat NH₄Cl, and extracted withether (3×15 mL). The organic layers were dried with MgSO₄, filtered, andconcentrated. Flash column chromatography (hexanes/ethyl acetate, 9:1)gave 0.0040 g (67%) of the α-alcohol 15C-(S) as a pale yellow oil and0.0006 g of β-alcohol 15C-(R).

EXAMPLE 36

[0243] Diol 15C″

[0244] The silyl ether 15 C-(S) (0.010 g, 0.012 mmol) was dissolved inHF•pyridine/pyridine/THF (1 mL). The solution was stirred at rt. for 2h, then diluted with Et₂O (1 mL), poured into a mixture of Et₂O/sat.NaHCO₃ (20 mL, 1:1), and extracted with Et₂O (4×10 mL). The Et₂Osolutions were washed with sat CuS0₄ (3×30 mL), sat NaHCO₃ (1×30 mL),brine (1×30 mL), dried with MgSO₄, filtered, and concentrated. Flashcolumn chromatography (hexanes/ethyl acetate, 9:1) gave 0.0066 g (93%)of the diol 15C″ as a pale yellow oil.

EXAMPLE 37

[0245] Alcohol 15C′″

[0246] To a solution of diol 15C″ (0.0066 g, 0.011 mmol) in 0.5 mL ofCH₂Cl₂ at −78° C. was added 2,6-lutidine (7 μL, 0.060 mmol) and TBSOTf(5 μL, 0.022 mmol). The resulting solution was stirred at −30° C. for0.5 h, then quenched with H₂O (5 mL), and extracted with Et₂O (4×10 mL).The ether solutions were washed with 0.5 M HCl (1×10 mL), sat'd NaHCO₃(1×10 mL), dried over MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 93:7) gave 0.0070 g (89%) of thealcohol 15C′″ as a pale yellow oil.

EXAMPLE 38

[0247] Ketone 16C

[0248] To a solution of alcohol 15C′″ (0.006 g, 0.0083 mmol) in 0.5 mLof CH₂Cl₂ at rt. was added Dess-Martin periodinane (0.030g, 0.071 mmol).After 1.25 h, another portion of Dess-Martin periodinane (0.025 mg,0.059 mmol) was added. The resulting solution was stirred at rt foradditional 0.75 h, treated with ether (1 mL) and sat'd Na₂S₂O₃/sat'dNaHCO₃ (2 mL, 1:1), poured into H₂O (20 mL), and extracted with ether(4×10 mL). The ether solution was washed with sat NaHCO₃ (1×20 mL),dried with MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 9:1) gave 0.0040 g (67%) of theketone 16C as a pale yellow oil.

EXAMPLE 39

[0249] Desoxyepothiolone B (2C)

[0250] To a solution of ketone 16C (0.004 g, 0.0056 mmol) in THF (0.35mL) was added HF•pyridine (0.25 mL) dropwise over 20 min. The solutionwas stirred at rt for 1.5 h, diluted with CHCl₃ (2 mL), poured intosat'd NaHCO₃/CHCl₃ (20 mL, 1:1) slowly, and extracted with CHCl₃ (4×10mL). The combined CHCl₃ layers were dried with MgSO₄, filtered, andconcentrated. Flash column chromatography (hexanes/ethyl acetate, 3:1)gave 0.0022 g (80%) of the desoxyepothilone B 2C as a pale yellow oil.

EXAMPLE 40

[0251] Epothilone B (2)

[0252] To a solution of desoxyepothilone B (0.0022 g, 0.0041 mmol) inCH₂Cl₂ (0.25 mL) at −50° C. was added dimethyldioxirane (0.1 mL, 0.0095mmol) dropwise. The resulting solution was stirred at −50° C. for 1 h.The dimethyldioxirane and solvent were removed by a stream of N₂. Theresidue was purified by flash column chromatography (hexanes/ethylacetate, 1:1) gave 0.0015 g (70%) of epothiolone B (2) as a pale yellowoil which was identical with an authentic sample in ¹H NMR, IR, massspectrum, and [α]_(D).

EXAMPLE 41

[0253] 8-Desmethylepothilone A

[0254] Crotylation Product

[0255] To a stirred mixture of potassium tert-butoxide (1.0 M soln inTHF, 50.4 mL, 50.4 mmol), THF (14 mL), and cis-2-butene (9.0 mL, 101mmol) at −78° C. was added n-BuLi (1.6 M, in hexanes, 31.5 mL, 50.4mmol). After complete addition of n-BuLi, the mixture was stirred at−45° C. for 10 min and then cooled to −78° C.(+)-B-Methoxydiisopinocampheylborane (19.21 g, 60.74 mmol) was thenadded dropwise in Et₂O (10 mL). After 30 min, BF₃•Et₂O (7.47 mL, 60.74mmol) was added followed by aldehyde 4D (9.84 g, 60.74 mmol) in THF (15mL) generating a viscous solution which could not be stirred. Themixture was shaken vigorously every 10 min to ensure homogeneity. After3 h at −78° C., the reaction was treated with 3N NaOH (36.6 mL, 110mmol) and 30% H₂O₂ (15 mL) and the solution brought to reflux for 1 h.The reaction was poured into Et₂O (300 mL) and washed with H₂O (100 mL),brine (30 mL) and dried over anhydrous MgSO₄. The crude material wasplaced in a bulb-to-bulb distillation apparatus to remove the ligandfrom the desired product. Heating at 80° C. at 2 mm Hg removed 90% ofthe lower boiling ligand.

[0256] Further purification of the alcohol 4D was achieved by flashchromatography on silica gel eluting with Et₂O in CH₂Cl₂ (2% - 4%) togive pure alcohol 4D as a clear oil. The erythro selectivty was >50:1 asjudged by ¹H NMR spectroscopy. The product was determined to be 87% eeby formation of the Mosher ester: IR (film): 3435, 2861, 1454, 1363,1099 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.34 (5H, m), 5.80 (1H, m), 5.09(1H, dd, J=1.6, 8.3 Hz), 5.04 (1H, d, J=1.6 Hz), 4.52 (2H, s), 3.51 (2H,t, J=5.8 Hz), 3.47 (1H, m) 2.27 (2H, m), 1.73 (3H, m), 1.42 (1H, m),1.04 (3H, d, J=6.9 Hz); ¹³C NMR (CDCl₃, 100 MHz), δ 141.1, 138.2, 128.3,127.6, 127.5, 115.0, 74.5, 72.9, 70.4, 43.7, 31.3, 26.5, 14.6.

EXAMPLE 42

[0257] TBS ether 5D

[0258] Alcohol 4D (5.00 g, 21.4 mmol) was dissolved in CH₂Cl₂ (150 mL)and 2,6-lutidine (9.97 mL, 85.6 mmol) was added. The mixture was cooledto 0° C. and TBSOTf (9.83 mL, 42.8 mmol) was slowly added. The reactionwas then warmed to rt. After 1 h, the reaction was poured into Et₂O (300mL) and washed once with 1 N HCl (50 mL), once with sat NaHCO₃ (50 mL),once with brine (30 mL) and dried over anhydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/diethyl ether(97:3) gave 6.33 g (85%) of pure olefin 5D as a clear oil: IR (film):1472, 1361, 1255, 1097, 1068 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.30 (5H,m), 5.81 (1H, m), 4.97 (1H, dd, J=1.4, 4.8 Hz), 4.94 (1H, d, J=1.1 Hz),3.51 (1H, q, J=5.1 Hz), 3.41 (2H, dt, J=2.1, 6.6 Hz), 2.27 (1H, q, J=5.5Hz), 1.68 (1h, m), 1.55 (1H, m), 1.41 (2H, m), 0.93 (3H, d, J=6.9 Hz),0.85 (9H, s), −0.01 (6H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 141.2, 138.6,128.3, 127.6, 127.4, 113.9, 75.6, 72.7, 70.6, 42.7, 30.1, 25.9, 25.4,18.1, 15.1, −4.3, −4.4.

EXAMPLE 43

[0259] Aldehyde 6D

[0260] The olefin 5 (4.00 g, 11.49 mmol) was dissolved in 1:1MeOH/CH₂Cl₂ (100 mL). Pyridine (4.0 mL) was then added and the mixturecooled to −78° C. Ozone was then bubbled through the reaction for 10minutes before the color turned light blue in color. Oxygen was thenbubbled through the reaction for 10 min. Dimethyl sulfide (4.0 mL) wasthen added and the reaction slowly warmed to rt. The reaction wasstirred overnight and then the volatiles were removed in vacuo.Purification by flash chromatography on silica gel eluting withhexanes/ethyl acetate (9:1) gave 3.31 g (82%) of the aldehyde 6D as aclear oil:

[0261] IR (film): 2856, 1727, 1475, 1361, 1253, 1102 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 9.76 (1H, s), 7.33 (5H, m), 4.50 (2H, s), 4.11 (1H,m), 3.47 (2H, m), 2.46 (1H, m), 1.50-1.70 (4H, band), 1.05 (3H, d, J=7.0Hz), 0.86 (9H, s), 0.06 (3H, s), 0.03 (3H, s); ¹³C NMR (CDCl₃, 100 MHz)δ 204.8, 138.3, 128.2, 127.4, 127.3, 72.7, 71.7, 69.9, 51.1, 31.1, 25.9,25.6, 17.8, 7.5, −4.4, −4.8.

EXAMPLE 44

[0262] Dianion Addition Product 7D

[0263] The tert-butyl isobutyrylacetate (0.653 g, 3.51 mmol) was addedto a suspension of NaH (60% in mineral oil, 0.188 g, 4.69 mmol) in THF(50 mL) at rt. After 10 min, the mixture was cooled to 0° C. After anadditional 10 min, n-BuLi (1.6 M in hexanes, 2.20 mL, 3.52 mmol) wasslowly added. After 30 min, the aldehyde 6D (1.03 g, 2.93 mmol) wasadded neat. After 10 min, the reaction was quenched with H₂O (10 mL) andextracted with Et₂O (2×75 mL). The combined organics were washed oncewith brine (30 mL) and dried over anhydrous MgSO₄. The crude reactionmixture contained a 15:1 ratio of diastereomers at C5. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(9:1→7:1) gave 0.723 g (47%) of the desired alcohol 7D as a clear oil:

[0264] IR (film): 3531, 2953, 1739, 1702, 1367, 1255, 1153 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 7.33 (5H, m), 4.49 (2H, s), 3.75 (1H, d, J=2.6 Hz),3.71 (1H, m), 3.62 (1H, d, J=16.0 Hz), 3.53 (1H, d, J=16.0 Hz), 3.44(2H, t, J=5.1 Hz), 2.70 (1H, d, J=2.6 Hz), 1.83 (1H, m), 1.55 (4H, m),1.46 (9H, s), 1.17 (3H, s), 1.11 (3H, s), 0.89 (9H, s), 0.82 (3H, d,J=7.0 Hz), 0.09 (6H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 208.9, 167.3, 138.4,128.3, 127.6, 127.5, 81.3, 79.5, 78.7, 72.8, 70.1, 52.4, 47.6, 35.8,30.6, 28.2, 25.9, 25.8, 22.6, 20.5, 17.9, 7.05, −4.0, −4.5.

EXAMPLE 45

[0265] Directed Reduction

[0266] To a solution of tetramethylammonium triacetoxyborohydride (1.54g, 5.88 mmol) in acetonitrile (4.0 mL) was added anhydrous AcOH (4.0mL). The mixture was stirred at rt for 30 min before cooling to −10° C.A solution of the ester 7D (0.200 g, 0.39 mmol) in acetonitrile (1.0 mL)was added to the reaction and it was stirred at -10° C. for 20 h.

[0267] The reaction was quenched with 1 N sodium-potassium tartrate (10mL) and stirred at rt for 10 min. The solution was then poured into satNaHCO₃ (25 mL) and neutralized by the addition of solid Na₂CO₃. Themixture was then extracted with EtOAc (3×30 mL) and the organics werewashed with brine (20 mL) and dried over anydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(4:1) gave 0.100 g (50%) of the diol as 10:1 ratio of diastereomericalcohols.

EXAMPLE 46

[0268] Monoprotection of the Diol

[0269] The diol (1.76 g, 3.31 mmol) was dissolved in CH₂Cl₂ (100 mL) andcooled to 0° C. 2,6-lutidine (12.2 mL, 9.92 mmol) was added followedbyTBSOTf (1.14 mL, 4.96 mmol) and the reaction slowly warmed to rt.After 1 h, the reaction was poured into Et₂O (300 mL) and washed oncewith 1 N HCl (50 mL), once with sat NaHCO₃ (50 mL), once with brine (30mL) and dried over anhydrous MgSO₄. Purification by flash chromatographyon silica gel eluting with hexanes/ethyl acetate (20:1→15:1) gave 2.03 g(95%) of the alcohol 8D as a clear oil, which was used as a mixture ofdiastereomers.

EXAMPLE 47

[0270] CS Ketone Formation

[0271] The alcohol 8D (2.03 g, 3.14 mmol) was dissolved in CH₂Cl₂ (50mL) and Dess-Martin periodinane (2.66 g, 6.28 mmol) was added. After 2h, a 1:1 mixture of sat'd NaHCO₃/sat Na₂S₂O₃ (20 mL) was added. After 10min, the mixture was poured into Et₂O (300 mL) and the organic layer waswashed with brine (30 mL) and dried over anhydrous MgSO₄. Purificationby flash chromatography on silica gel eluting with hexanes/ethyl acetate(15:1) gave 1.85 g (91%) of the ketone (benzyl ether) as a clear oil,which was used as a mixture of diastereomers.

EXAMPLE 48

[0272] Debenzylation

[0273] The ketone (benzyl ether) (1.85 g, 2.87 mmol) was dissolved inEtOH (50 mL), and Pd(OH)₂ (0.5 g) was added. The mixture was thenstirred under an atmosphere of H₂. After 3 h, the reaction was purgedwith N₂ and then filtered through a pad of celite rinsing with CHCl₃(100 mL). Purification by flash chromatography on silica gel elutingwith ethyl acetate in hexanes (12%→15%) gave 1.43 g (90%) of thediastereomeric alcohols as a clear oil. The C3 diastereomers wereseparated by flash chromatography on TLC-grade SiO₂ eluting with ethylacetate in hexanes (15%):

[0274] Alpha isomer: IR (film): 3447, 1732, 1695, 1254, 1156 cm⁻¹; ¹HNMR (CDCl₃, 400 MHz) δ 4.24 (1H, dd, J=3.6, 5.8 Hz), 3.83 (1H, m), 3.53(1H, m), 3.06 (1H, t, J=7.1 Hz) 2.36 (1H, dd, J=3.6, 17.2 Hz), 2.12 (1H,dd, J=3.9, 17.2 Hz), 1.68 (1H, t, J=5.4Hz), 1.54 (2H, m), 1.41 (1H, m),1.37 (9H, s), 1.31 (1H, m), 1.16 (3H, s), 1.02 (3H, s), 0.99 (3H, dJ=6.8 Hz), 0.84 (9H, s), 0.81 (9H, s), 0.05 (3H, s), 0.01 (6H, s), −0.01(3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.7, 171.3, 80.57, 73.5, 73.1,63.0, 53.4, 26.8, 41.2, 32.1, 28.1, 28.0, 26.0, 25.9, 23.1, 19.8, 18.1(overlapping), 15.3, −4.0, −4.3 (overlapping), −4.8.

[0275] Beta isomer: IR (film): 3442, 2857, 1732, 1700, 1472, 1368, 1255cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 4.45 (1H, t, J=5.3 Hz), 3.86 (1H, m),3.52 (2H, q, J=5.9 Hz), 3.01 (1H, m), 2.28 (1H, dd, J=4.3, 17.1 Hz),2.16 (1H, dd, J=5.5, 17.1 Hz), 1.67 (1H, t, J=5.6 Hz), 1.56 (2H, m),1.44 (1H, m), 1.37 (9H, s), 1.34 (1H, m), 1.13 (3H, s) 0.97 (3H, d,J=7.4 Hz), 0.96 (3H, s), 0.83 (9H, s), 0.79 (9H, s), 0.01 (3H, s), 0.00(6H, s), −0.07 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.1, 171.2, 80.6,73.5, 72.1, 62.9, 63.9, 46.4, 41.2, 32.0, 28.1, 28.0, 26.0, 25.9, 21.5,19.5, 18.2, 18.1, 15.8, −4.0, −4.3, −4.4, −4.7.

EXAMPLE 49

[0276] Aldehyde Formation

[0277] DMSO (0.177 mL, 2.50 mmol) was added to a mixture of oxalylchloride (0.11 mL, 1.25 mmol) in CH₂Cl₂ (15 mL) at −78° C. After 10 min,the alcohol (0.531 g, 0.96 mmol) was added in CH₂Cl₂ (4 mL). After 20min, TEA (0.697 mL, 5.00 mmol) was added to the reaction followed bywarming to rt. The reaction was then poured into H₂O (50 mL) andextracted with Et₂O (3×50 mL). The organics were washed once with H₂O(30 mL), once with brine (30 mL) and dried over anhydrous MgSO₄. Thealdehyde was used in crude form.

EXAMPLE 50

[0278] Wittig Olefination to give 9D

[0279] NaHMDS (1.0 M soln in THF, 1.54 mL, 1.54 mmol) was added to asuspension of methyl triphenylphosphonium bromide (0.690 g, 1.92 mmol)in THF (20 mL) at 0° C. After 1 h, the crude aldhyde (0.96 mmol) wasadded in THF (5 mL). After 15 min at 0° C., H₂O (0.1 mL) was added andthe reaction poured into hexanes (50 mL). This was filtered through aplug of silica gel eluting with hexanes/Et₂O (9:1, 150 mL). The crudeolefin 9D was further purified by flash chromatography on silica geleluting with ethyl acetate in hexanes (5%) to give 0.437 g (83% for twosteps) of the olefin 9D as a clear oil: IR (film): 2857, 1732, 1695,1472, 1368, 1255, 1156cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 5.72 (1H, m), 4.91(2H, m), 4.25 (1H, dd, J=3.9, 5.4 Hz), 3.81 (1H, m), 3.05 (1H, m), 2.38(1H, dd, J=7.9, 17.2 Hz), 2.12 (1H, dd, J=6.6, 17.2 Hz), 2.04 (2H, q,J=7.5 Hz), 1.47 (1H, m), 1.39 (9H, s), 1.34 (1H, m), 1.20 (3H, s), 1.00(3H, s), 3.00 (3H, d, J=6.7 Hz), 0.85 (9H, s), 0.83 (9H, s), 0.07 (3H,s), 0.00 (6H, s), −0.05 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.5,172.1, 137.9, 114.0, 80.4, 74.0, 73.0, 53.0, 46.9, 41.3, 35.1, 29.0,28.1, 26.0, 25.9, 22.8, 20.2, 18.2 (overlapping), 14.9, −4.1, −4.2,−4.3, −4.8.

EXAMPLE 51

[0280] TBS Ester 10D

[0281] The olefin 9D (0.420 g, 0.76 mmol) was dissolved in CH₂Cl₂ (15mL) and treated successively with 2,6-lutidine (1.33 mL, 11.4 mmol) andTBSOTf (1.32 mL, 5.73 mmol). After 7 h, the reaction was poured intoEt₂O (100 mL) and washed successively with 0.2N HCl (25 mL), brine (20mL) and dried over anhydrous MgSO₄. The residue was purified by flashchromatography on a short pad of silica gel with fast elution withhexanes/ethyl acetate (20:1) to give the TBS ester 10D as a clear oil.The purification must be done quickly to avoid hydrolysis of the silylester: IR (film): 2930, 1721, 1695, 1472, 1254, 1091 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 5.73 (1H, m), 4.91 (2H, m), 4.25 (1H, dd, J=3.8, 5.4Hz) 3.80 (1H, q, J=6.8 Hz), 3.06 (1H, m), 2.50 (1H, dd, J=3.7, 17.3 Hz),2.19 (1H, d, J=5.7, 17.3 Hz), 2.04 (2H, dd, J=7.6, 15.3 Hz), 1.49 (1H,m), 1.36 (1H, m), 1.21 (3H, s), 1.00 (3H, d, J=6.8 Hz), 0.88 (9H, s),0.85 (9H, s), 0.83 (9H, s), 0.22 (3H, s), 0.22 (3H, s), 0.21 (3H, s),0.06 (3H, s), 0.01 (6H, s), −0.05 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ217.3, 172.3, 138.5, 114.4, 74.5, 73.0, 53.2, 46.9, 41.8, 35.1, 29.0,26.0, 25.7, 25.5, 22.8, 20.4, 18.2, 18.1, 17.5, 14.9, −2.9, −4.0, −4.2,−4.3, −4.8, −4.9.

EXAMPLE 52

[0282] Suzuki Coupling

[0283] The acetate acid 13D was purified by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (7:1→4:1). This wasfurther purified by preparative-TLC eluting with hexanes/ethyl acetate(2:1) to remove unreacted vinyl iodide 12D from the acetate acid 13D.Isolated yield of the acid was 0.297 g (62% based on 90% purity withborane residues).

EXAMPLE 53

[0284] Hydrolysis of Acetate Acid 13D

[0285] The acetate 13D (0.220 g, 0.297 mmol) was dissolved in MeOH/H₂O(2:1, 15 mL) and K₂CO₃ (0.300 g) was added. After 3 h, the reaction wasdiluted with sat NH₄Cl (20 mL) and extracted with CHCl₃ (5×20 mL). Thehydroxy-acid 14D was purified by flash chromatography on silica geleluting with hexanes/ethyl acetate (4:1→2:1) to give 0.146 g (70%) ofthe pure hydroxy acid 14D. IR (film): 3510-2400, 1712, 1694, 1471, 1254,1093 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.96 (1H, s), 6.66 (1H, s), 5.55(1H, m), 5.38 (1H, m), 4.38 (1H, dd, J=3.4, 6.1 Hz), 4.19 (1H, t, J=7.5Hz), 3.84 (1H, m), 3.05 (1H, t, J=7.0 Hz), 2.72 (3H, s), 2.49 (1H, dd,J=3.2, 16.4 Hz), 2.42 (2H, m), 2.33 (1H, dd, J=6.2, 16.4 Hz), 2.07 (2H,m), 2.02 (3H, s), 1.33 (4H, m), 1.19 (3H, s), 1.14 (3H, s), 1.06 (3H, d,J=6.7 Hz), 0.89 (9H, s), 0.88 (9H, s), 0.11 (3H, s), 0.07 (3H, s) 0.04(6H, s); ¹³C NMR(CDCl₃, 100 MHz) δ 217.8, 176.6, 164.9, 152.5, 141.7,132.9, 125.0, 119.0, 115.3, 73.5, 73.3, 53.4, 47.0, 40.1, 35.8, 33.2,29.8, 27.4, 26.0, 25.9, 24.5, 19.0, 18.1, 15.2, 14.3, −4.0, −4.2, −4.2,−4.7.

EXAMPLE 54

[0286] Macrolactonization

[0287] DCC (0.150 g, 0.725 mmol), 4-DMAP (0.078 g, 0.64 mmol) and4-DMAP•HCl (0.110 g, 0.696 mmol) were dissolved in CHCl₃ (80 mL) at 80°C. To this refluxing solution was added by syringe pump the hydroxy acid14D (0.020 g, 0.029 mmol) and DMAP (0.010 g) in CHCl₃ (10 mL) over 20 h.The syringe needle was placed at the base of the condensor to ensureproper addition. After 20 h, the reaction was cooled to 50° C. and AcOH(0.046 mL, 0.812 mmol) was added. After 2 h, the reaction was cooled tort and washed with sat NaHCO₃ (30 mL), brine (30 mL) and dried overanhydrous Na₂SO₄. The lactone 15D was purified by flash chromatographyon silica gel eluting with hexanes/ethyl acetate (20:1→15:1) to give0.014 g (75%): IR (film): 2929, 1741, 1696, 1254, 1097 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 6.95 (1H, s), 6.55 (1H, s), 5.48 (1H, m), 5.37 (1H,m), 5.16 (1H, d, J=9.8 Hz), 4.17 (1H, d, J=8.3 Hz), 4.07 (1H, t, J=7.2Hz), 3.02 (1H, t, J=7.2 Hz), 2.77 (1H, m), 2.70 (3H, s), 2.64 (2H, m),2.29 (1H, m), 2.15 (1H, m), 2.12 (3H, s), 1.92 (1H, m), 1.71 (1H, m),1.44 (2H, m), 1.26 (1H, m), 1.17 (3H, s), 1.12 (3H, s) 1.11 (3H, d,J=7.0 Hz), 0.91 (9H, s), 0.85 (9H, s), 0.09 (3H, s), 0.06 (6H, s), −0.04(3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 215.2, 171.9, 164.5, 152.5, 138.0,133.5, 123.8, 120.0, 116.7, 79.4, 76.2, 72.5, 53.5, 47.4, 39.9, 34.5,31.9, 31.5, 30.2, 27.7, 26.1, 25.9, 24.1, 23.8, 23.1, 22.6, 19.2, 18.5,18.2, 16.3, 14.9, 14.1, −3.7, −4.2, −4.7, −5.2.

EXAMPLE 55

[0288] Desmethyldesoxyepothilone A (16D)

[0289] To the lactone 15D (0.038 g, 0.056 mmol) in THF (2.0 mL) wasadded HF-pyridine (1.0 mL). After 2 h, the reaction was poured into satNaHCO₃ (30 mL) and extracted with CHCl₃ (5×20 mL). The organics weredried over Na₂SO₄. The crude diol 16D was purified by flashchromatography on silica gel eluting with hexanes/ethyl acetate(3:1→2:1) to give 0.023 g (89%): IR (film): 3501, 2933, 1734, 1684,1290, 1248, 1045 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.95 (1H, s), 6.59 (1H,s), 5.40 (2H, m), 5.53 (1H, dd, J=1.4, 9.5 Hz), 4.38 (1H, bd, J=11.1Hz), 3.78 (1H, t, J=6.9 Hz), 3.59 (1H, bs), 3.47 (1H, s), 2.99 (1H, q,J=7.0 Hz), 2.68 (3H, s), 2.66 (1H, m), 2.46 (1H, dd, J=11.4, 14.4 Hz),2.26 (1H, dd, J=2.2, 14.4 Hz), 2.22 (1H, m), 2.06 (3H, s), 1.96 (1H, m),1.49 (3H, m), 1.35 (3H, m), 1.30 (3H, s), 1.15 (3H, d, J=6.9 Hz), 1.06(3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 221.5, 170.3, 165.1, 151.8, 139.1,132.8, 125.2, 119.1, 115.5, 78.4, 72.5, 70.8, 53.8, 42.7, 39.6, 32.3,31.8, 28.3, 26.8, 24.8, 23.1, 19.0, 17.2, 16.0, 11.1.

EXAMPLE 56

[0290] Epoxide Formation

[0291] Diol 16D (0.008 g, 0.017 mmol) was dissolved in CH₂Cl₂ (1.0 mL)and cooled to −60° C. Dimethyldioxirane (0.06 M, 0.570 mL, 0.0034 mmol)was then slowly added. The reaction temperature was slowly warmed to−25° C. After 2 h at −25° C., the volatiles were removed from thereaction at −25° C. under vacuum. The resulting residue was purified byflash chromatography on silica gel eluting with MeOH in CH₂Cl₂ (1% →2%)to give a 1.6:1 mixture of cis -epoxides 3D and the diastereomericcis-epoxide (0.0058 g, 74%). The diastereomeric epoxides were separatedby preparative-TLC eluting with hexanes/ethyl acetate (1:1) after 3elutions to give pure diastereomers:

[0292] Beta epoxide 3D: IR (film): 3458, 2928, 1737, 1685, 1456, 1261,1150, 1043, 1014 cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ 7.01 (1H, s), 6.56(1H, s), 5.35 (1H, dd, J=2.3, 9.6 Hz), 4.30 (1H, dd, J=3.0, 5.7 Hz),3.85 (1H, m), 3.81 (1H, d, J=5.7 Hz), 3.42 (1H, dd, J=2.0 Hz), 3.03 (1H,q, J=6.8 Hz), 2.97 (1H, m), 2.88 (1H, m), 2.67 (3H, s), 2.46 (1H, dd,J=9.0, 14.5 Hz), 2.33 (1H, dd, J=2.6, 14.5 Hz), 2.13 (1H, dt, J=3.0,15.0 Hz), 2.08 (3H, s), 1.82 (1H, m), 1.52 (6H, m), 1.41 (1H, m), 1.33(3H, s), 1.21 (4H, m), 1.12 (3H, d, J=7.0 Hz), 1.06 (3H, s); ¹³C NMR(CD₂Cl₂, 125 MHz) δ 221.9, 170.6, 165.6, 152.2, 138.3, 120.2, 116.6,77.3, 73.4, 69.9, 57.7, 55.3, 43.7, 39.7, 32.6, 32.0, 29.8, 27.2, 25.7,24.7, 22.5, 19.2, 19.0, 15.6, 15.6, 11.5;

[0293] Alpha epoxide: IR (film): 3439, 2918, 1735, 1684, 1455, 1262,1048, 1014 cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ 7.02 (1H, s), 6.56 (1H, s),5.62 (1H, d, J=8.1 Hz), 4.33 (1H, dd, J=2.7, 11.0 Hz), 3.85 (1H, t,J=5.9 Hz), 3.27 (1H, d, J=5.3 Hz), 3.11 (1H, m), 3.07 (1H, d, J=7.0 Hz),3.04 (1H, s), 2.87 (1H, m), 2.68 (3H, s), 2.46 (1H, dd, J=11.1, 14.1Hz), 2.35 (1H, dd, J=2.3, 14.1 Hz), 2.11 (3H, s), 2.06 (1H, ddd, J=1.9,4.5, 15.1 Hz), 1.87 (1H, m), 1.52 (6H, m), 1.38 (2H, m), 1.29 (3H, s),1.08 (3H, d, J=6.9 Hz), 1.03 (3H, s); ¹³C NMR (CD₂Cl₂, 125 MHz) δ 222.1,170.2, 165.3, 152.5, 137.6, 119.7, 116.7, 76.7, 72.9, 70.6, 57.1, 55.1,44.7, 40.0, 32.1, 31.4, 30.0, 26.6, 25.5, 24.7, 21.3, 19.3, 18.7, 15.7,11.5.

EXAMPLE 57

[0294] Experimental Data for C-12Hydroxy Epothilone Analogs

[0295] Propyl Hydroxy Compound 43

[0296]¹H NMR (CDCl₃, 400 MHz) d 6.96 (1H, s), 6.59 (1H, s), 5.16-5.23(2H, band), 4.28 (1H, m), 3.72 (1H, m), 3.63 (2H, t, J=6.3 Hz), 3.17(1H, dq, J=2.2, 0.5 Hz), 3.02 (1H, s), 2.70 (3H, s), 2.65 (2H, m), 2.46(1H, dd, J=10.9, 14.6 Hz), 2.29 (2H, m), 1.98-2.09 (6H, band), 1.60-1.91(6H, band), 1.35 (3H, s), 1.18 (3H, d, J=6.8 Hz), 1.07 (3H, s), 1.01(3H, d, J=7.1 Hz); ¹³C NMR (CDCl₃, 100 MHz) d 220.69, 170.29, 165.00,151.81, 141.63, 138.93, 120.64, 118.81, 115.52, 78.53, 77.23, 73.93,71.85, 62.26, 53.63, 41.57, 39.54, 37.98, 32.33, 32.14, 31.54, 30.75,29.67, 25.27, 22.89, 18.92, 17.67, 15.98, 15.74, 13.28; MS e/m 536.2,calc 535.29.

[0297] Hydroxy Methyl Compound 46

[0298]¹H NMR (CDCl₃, 400 MHz) d 6.97 (1H, s), 6.63 (1H, s), 5.43 (1H,dd, j =5.7, 9.1 Hz), 5.24 (1H, d, J=7.4 Hz), 4.31 (1H, d, J=9.7 Hz),4.05 (2H, dd, J=7.3, 31.0 Hz), 3.87 (1H, bs), 3.69 (1H, bs), 3.17 (1H,dd, J=2.0, 6.9 Hz), 3.03 (1H, s), 2.69 (3H, s), 2.63 (1H, m), 2.45 (1H,dd, J=11.2, 14.6 Hz), 2.37 (1H, m) 2.25 (2H, m), 2.11 (1H, m), 2.05 (3H,s), 1.78 (1H, m), 1.70 (1H, m), 1.35 (3H, s), 1.34 (2H, m), 1.29 (1H,m), 1.18 (3H, d, J=6.8 Hz), 1.06 (3H, s), 1.00 (3H, d, J=7.0 Hz); ¹³CNMR (CDCl₃, 100 MHz) d 220.70, 170.16, 165.02, 151.63, 141.56,138.41,121.33, 118.65, 115.33, 77.74, 77.25, 74.11, 71.37, 65.75, 53.86, 41.52,39.52, 37.98, 31.46, 27.70, 25.10, 22.86, 18.74, 17.20,16.17,15.63,13.41.

[0299] Discussion

[0300] Total Synthesis of (−)-Epothilone A.

[0301] The first known method for preparing epothilone A (1) is providedby this invention. Carbons 9 through 11 insulate domains of chiralityembracing carbons 3 through 8 on the acyl side of the macrolactone, andcarbons 12 through 15 on the alkyl side. Transmitting stereochemicalinformation from one of the segments to the other is unlikely. Thus, theapproach taken deals with the stereochemistry of each segmentindividually. In the acyl segment, this strategy required knowledge ofboth the relative and absolute configurations of the“polypropionate-like” network. In the alkyl segment, two possibilitiesemerge. In one instance, the C12-C13 epoxide would be included in theconstruct undergoing merger with the acyl related substructure. In thatcase it would be necessary to secure the relative stereochemicalrelationship of carbons 15, 13 and 12. It was necessary to consider thethe possibility that the epoxide would be deleted from the alkyl-sidemoiety undergoing coupling. This approach would only be feasible if theepoxide could be introduced with acceptable stereocontrol after closureof the macrocycle. The synthesis of compound 4, which contains most ofthe requisite stereochemical information required for the acyl fragment,is described above. This intermediate is prepared by a novel oxidativelyinduced solvolytic cleavage of the cyclopropanopyran 3. Also describedabove is a construct containing the alkyl side coupling partnerembodying the absolute and relative stereochemistry at carbons 15, 13and 12, which differs from the alternative approach set forth below.

[0302] In considering the union of the alkyl and acyl domains, severalpotential connection sites were available. At some point, an acylationwould be required to establish an ester (or lactone) bond (see boldarrow 2). Furthermore, an aldol construction was required to fashion aC2-C3 connection. Determining the exact timing of this aldol steprequired study. It could be considered in the context of elongating theC3-C9 construct to prepare it for acylation of the C-15 hydroxyl.Unexpectedly, it was discovered that the macrolide could be closed bv anunprecedented macroaldolization. (For a previous instance of a ketoaidehyde macroaldolization, see: C. M. Hayward, et al., J. Am. Chem.Soc., 1993, 115, 9345.) This option is implied by bold arrow 3 in FIG.1(A).

[0303] The first stage merger of the acyl and alkyl fragments (see boldarrow 1) posed a difficult synthetic hurdle. It is recognized in the art(P. Bertinato, et al., J. Org. Chem., 1996, 61, 8000; vide infra) thatsignificant resistance is encountered in attempting to accomplish bondformation between carbons 9 and 10 or between carbons 10 and 11, whereinthe epoxide would be included in the alkyl coupling partner. Thesecomplications arose from unanticipated difficulties in fashioning acyland alkyl reactants with the appropriate complementarity for mergeracross either of these bonds. An initial merger between carbons 11 and12 was examined. This approach dictated deletion of the oxirane linkagefrom the O-alkyl coupling partner. After testing several permutations,generalized systems 5 and 6 were examined to enter the first stagecoupling reaction. The former series was to be derived from intermediate4. A de novo synthesis of a usable substrate corresponding togeneralized system 5 would be necessary (FIG. 1(B)).

[0304] The steps leading from 4 to 11 are shown in Scheme 2. Protectionof the future C-7 alcohol (see compound 7) was followed by cleavage ofthe benzyl ether and oxidation to aldehyde 8. Elongation of the aldehydeto the terminal allyl containing fragment 10 proceeded through end ether9 (mixture of E and Z geometrical isomers). Finally, the dithianelinkage was oxidatively cleaved under solvolytic trapping conditions,giving rise to specific coupling component 11. G. Stork; K. Zhao,Tetrahedron Lett. 1989, 30, 287.

[0305] The synthesis of the alkyl fragment started with commerciallyavailable (R)-glycidol 12 which was converted, via its THP derivative13, to alcohol 14. After cleavage of the tetrahydropyran blocking group,the resultant alcohol was smoothly converted to the methyl ketone 15, asshown. The latter underwent an Emmons-type homologation with phosphineoxide 16. D. Meng et al., J. Org. Chem., 1996, 61, 7998. This Emmonscoupling provided a ca. 8:1 mixture of olefin stereoismoers in favor oftrans-17. The resultant alkyne 17 was then converted, via compound 18 toZ-iodoalkene 19 (see FIG. 4(A)). E. J. Corey et al., J. Am. Chem. Soc.,1985, 107, 713.

[0306] The critical first stage coupling of the two fragments wasachieved by a B-alkyl Suzuki carbon-carbon bond construction. N. Miyauraet al., J. Am. Chem. Soc., 1989, 111, 314; N. Miyaura and A. Suzuki,Chem. Rev., 1995, 95, 2457. Thus, hydroboration of the pre-acyl fragment11 was accomplished by its reaction with 9-BBN. The resultant mixedborane cross-coupled to iodoolefin 19, under the conditions indicated,to give 20 in 71% yield. (FIG. 4(B)) Upon cleavage of the acetal,aldehyde 21 was in hand.

[0307] The availability of 21 permitted exploration of the strategy inwhich the methyl group of the C-1 bound acetoxy function would serve asthe nucleophilic component in a macroaldolization. Cf. C. M. Hayward etal., supra. Deprotonation was thereby accomplished with potassiumhexamethyldisilazide in THF at −78° C. Unexpectedly, these conditionsgive rise to a highly stereoselective macroaldolization, resulting inthe formation of the C-3 (S)-alcohol 22, as shown. The heavypreponderance of 22 was favored when its precursor potassium aldolate isquenched at ca. 0° C. When the aldolate was protonated at lowertemperature, higher amounts of the C-3 (R) compound were detected. Infact, under some treatments, the C-3 (R) epimer predominates. It istherefore possible to generate highly favorable C-3(R):C-3(S) ratios inanalytical scale quenches. In preparative scale experiments, the ratioof 22 to its C-3 epimer is 6:1.

[0308] With compound 22 in ready supply, the subgoal of obtainingdesoxyepothilone (23) was feasible. This objective was accomplished byselective removal of the triphenylsilyl (TPS) group in 22, followed,sequentially, by selective silylation of the C-3 alcohol, oxidation ofthe C-5 alcohol, and, finally, fluoride-induced cleavage of the twosilyl ethers.

[0309] Examination of a model made possible by the published crystalstructure of epothilone (Höfle et al., supra), suggested that theoxirane is disposed on the convex periphery of the macrolide. Oxidationof 23 was carried out with dimethyl dioxirane under the conditionsshown. The major product of this reaction was (−)epothilone A (1), theidentity of which was established by nmr, infrared, mass spectral,optical rotation and chromotaraphic comparisons with authentic material.Höfle et al., supra. In addition to epothilone A (1), small amounts of adiepoxide mixture, as well as traces of the diastereomeric cis C12-C13monoepoxide (≧20:1 ) were detected.

[0310] The method of synthesis disclosed herein provides workable,practical amounts of epothilone A. More importantly, it provides routesto congeners, analogues and derivatives not available from the naturalproduct itself.

[0311] Studies Toward a Synthesis of Epothilone A: Use of HydropyranTemplates For the Management of Acyclic Stereochemical Relationships

[0312] The synthesis of an enantiomerically pure equivalent of thealkoxy segment (carbons 9-15) was carried out in model studies. The keyprinciple involves transference of stereochemical bias from an(S)-lactaldehyde derivative to an emerging dihydropyrone. The latter, onaddition of the thiazole moiety and disassembly, provides the desiredacyclic fragment in enantiomerically pure form.

[0313] Various novel structural features of the epothilones make theirsynthesis challenging. The presence of a thiazole moiety, as well as acis epoxide, and a geminal dimethyl grouping are key problems to beovercome. An intriguing feature is the array of three contiguousmethylene groups which serves to insulate the two functional domains ofthe molecules. The need to encompass such an achiral “spacer element”actually complicates prospects for continuous chirality transfer andseems to call for a strategy of merging two stereochemically committedsubstructures. The present invention provides a synthesis of compound 4A(FIG. 14), expecting that, in principle, such a structure could beconverted to the epothilones themselves, and to related screeningcandidates.

[0314] The identification of compound 4A as a synthetic intermediateserved as an opportunity to illustrate the power of hydropyran matricesin addressing problems associated with the control of stereochemistry inacyclic intermediates. The synthesis of dihydropyrones was previouslydisclosed through what amounts to overall cyclocondensation of suitablyactive dienes and aldehydic heterodienophiles. Danishefsky, S. J.Aldrichimica Acta, 1986, 19, 59. High margins of steroselectivity can berealized in assembling (cf. 5A+6A→7A) such matrices (FIG. 13). Moreover,the hydropyran platforms service various stereospecific reactions (seeformalism 7A→8A). Furthermore, the products of these reactions areamenable to ring opening schemes, resulting in the expression of acyciicfragments with defined stereochemical relationships (cf. 8A→9A).Danishefsky, S. J. Chemtracts, 1989, 2, 273.

[0315] The present invention provides the application of two such routesfor the synthesis of compound 4A. Route 1, which does not per se involvecontrol of the issue of absolute configuration, commences with the knownaldehyde 10A. Shafiee, A., et al., J. Heterocyclic Chem., 1979, 16,1563; Schafiee, A.; Shahocini, S. J. Heterocyclic Chem., 1989, 26,1627.Homologation, as shown, provided enal 12A. Cyclocondensation of 12Awith the known diene (Danishefsky, S. J.; Kitahara, T. J. Am. Chem.Soc., 1974, 96, 7807), under BF₃ catalysis, led to racemic dihydropyrone13A. Reduction of 13A under Luche conditions provided compound 14A.Luche, J.-L. J. Am. Chem. Soc., 1978, 100, 2226. At this point it wasfeasible to take advantage of a previously introduced lipase methodologyfor resolution of glycal derivatives through enzymatically mediatedkinetic resolution. Berkowitz, D. B. and Danishefsky, S. J. TetrahedronLett., 1991, 32, 5497; Berkowitz, D. B.; Danishefsky, S. J.; Schulte, G.K. J. Am. Chem. Soc., 1992, 114, 4518. Thus, carbinol 14A was subjectedto lipase 30, in the presence of isopropenyl acetate, following theprescriptions of Wong (Hsu, S.-H., et al., Tetrahedron Lett., 1990, 31,6403)to provide acetate 15A in addition to the enantiomerically relatedfree glycal 16A. Compound 15A was further advanced to the PMB protectedsystem 17A. At this juncture, it was possible to use another reactiontype previously demonstrated by the present inventors. Thus, reaction of17A with dimethyldioxirane (Danishefsky, S. J.; Bilodeau, M. T. Angew.Chem. Int. Ed. Engl., 1996, 35, 1381) generated an intermediate(presumably the corresponding glycal epoxide) which, upon treatment withsodium metaperiodate gave rise to aldehyde formate 18A. Allylation of18A resulted in the formation of carbinol 19A in which the formate esterhad nicely survived. (For a review of allylations, see: Yamamoto, Y.;Asao, N. Chem. Rev. 1993, 93, 2207.) However, 19A was accompanied by itsanti stereoisomer (not shown here) [4:1]. Mesylation of the secondaryalcohol, followed by deprotection (see 19A→20A) and cyclization, asindicated, gave compound 4A.

[0316] In this synthesis, only about half of the dihydropyrone wassecured through the process of kinetic resolution. While, in theory,several of the synthetic stratagems considered contemplate use of eachenantiomer of 15A to reach epothilone itself, another route was soughtto allow for full enantiomeric convergence. The logic of this route isthat the chirality of a “dummy” asymmetric center is communicated to theemerging pyran following previously established principles of tunablediastereoselection in the cyclocondensation reaction. (Danishefsky,supra) Cyclo-condensation of lactaldehyde derivative 21A (Heathcock, C.H., et al., J. Org. Chem., 1980, 45, 3846) with the indicated diene,under ostensible chelation control, afforded 22A. The side chain ethercould then be converted to the methyl ketone 25A as shown (see22A→23A→24A→25A). Finally, an Emmons condensations (for example, see:Lythgoe, B., et al., Tetrahedron Lett., 1975, 3863; Toh, H. T.; Okamura,W. H. J. Org. Chem., 1983, 48, 1414; Baggiolini, E. G., et al., J. Org.Chem., 1986, 51, 3098) of 25A with the phoshphine oxide 26A wastransformed to phosphine oxide 26A according to the procedure describedin Toh, supra) as shown in FIG. 15 gave rise to 27A. (The known2-methyl-4-chloromethylthiazole (see Marzoni, G. J. Heterocyclic Chem.,1986,23, 577.) A straightforward protecting group adjustment thenafforded the previously encountered 17A. This route illustrates theconcept of stereochemical imprinting through a carbon center whicheventually emerges in planar form after conferring enantioselection tosubsequently derived stereocenters. The use of the dihydropyrone basedlogic for securing the stereochemical elements of the epothilones, aswell as the identification of a possible strategy for macrocyclizationwill be described in the following section.

[0317] Studies Toward a Synthesis of Epothilone A: SterocontrolledAssembly of the Acyl Region and Models for Macrocyclization

[0318] Ring-forming olefin metathesis has been employed to construct16-membered ring congeners related to epothilone A. A stereospecificsynthesis of the C3-C9 sector of the acyl fragment was achieved byexploiting a novel oxidative opening of a cyclopropanated glycal.

[0319] Disclosed in the previous section is a synthesis of the “alkoxy”segment of epothilone (1) (see compound 2B, FIG. 7) encompassing carbons10 to 21. In this section the synthesis of another fragment encoding thestereochemical information of acyl section carbons 3 to 9. It wasenvisioned that the aldehydo center (C₃) of the formal target 3B wouldserve as an attachment site to a nucleophilic construct derived fromcompound 2B (requiring placement of a 2 carbon insert, as suggested inFIG. 7), through either inter- or intramolecular means. In such acontext, it would be necessary to deal independently with thestereochemistry of the secondary alcohol center eventually required atC₃. One of the interesting features of system 3B is the presence ofgeminal methyl groups at carbon 4 (epothilone numbering). Again, use ismade of a dihydropyran strategy to assemble a cyclic matrixcorresponding, after appropriate disassembly, to a viable equivalent ofsystem 3B. The expectation was to enlarge upon the dihydropyran paradigmto include the synthesis of gem-dimethyl containing cyclic and acyclicfragments. The particular reaction type for this purpose is generalizedunder the heading of transformation of 4B→5B (see FIG. 7). Commitment asto the nature of the electrophile E is avoided. Accordingly, thequestion whether a reduction would or would not be necessary in goingfrom structure type 5B to reach the intended generalized target 3B isnot addressed.

[0320] The opening step consisted of a stereochemically tuneable versionof the diene-aldehyde cyclocondensation reaction (FIG. 8; Danishefsky,S. J., Aldrichimica Acta, 1986, 19, 59), in this instance drawing uponchelation control in the merger of the readily availableenantiomerically homogenous aldehyde 6B with the previously known diene7B. Danishefsky, S. J., et al., J. Am. Chem. Soc. 1979, 101, 7001.Indeed, as precedent would have it, under the influence of titaniumtetrachloride there was produced substantially a single isomer shown ascompound 8B. In the usual and stereochemically reliable way(Danishefsky, S. J., Chemtracts Org. Chem. 1989, 2, 273), thedihydropyrone was reduced to the corresponding glycal, 9B. At thispoint, it was feasible to utilize a directed Simmons-Smith reaction forthe conversion of glycal 9B to cyclopropane 10B. Winstein, S.;Sonnenberg, J. J. Am. Chem. Soc., 1961, 83, 3235; Dauben, W. G.;Berezin, G. H. J. Am. Chem. Soc., 1963, 85, 468; Furukawa, J., et al.,Tetrahedron, 1968, 24, 53; For selected examples, see Soeckman, R. K.Jr.: Charette, A. B.; Asberom, T.; Johnston, B. H. J. Am. Chem. Soc.,1991, 113, 5337; Timmers, C. M.; Leeuwenurgh, M. A.; Verheijen, J. C.;Van der Marel, G. A.; Van Boom, J. H. Tetrahedron: Asymmetry, 1996, 7,49. This compound is indeed an interesting structure in that itcorresponds in one sense to a cyclopropano version of a C-glycoside. Atthe same time, the cyclopropane is part of a cyclopropylcarbinyl alcoholsystem with attendant possibilities for rearrangement. Wenkert, E., etal., J. Amer. Chem. Soc., 1970, 92, 7428. It was intended to cleave theC-glycosidic bond of the cyclopropane in a fashion which would elaboratethe geminal methyl groups, resulting in a solvent-derived glycoside withthe desired aidehyde oxidation state at C-3 (see hypothesizedtransformation 4B→5B, FIG. 7). In early efforts, the non-oxidativeversion of the projected reaction (i.e. E⁺=H⁺) could not be reduced topractice. Instead, products clearly attributable to the ring expandedsystem 11 were identified. For example, exposure of 10B to acidicmethanol gave rise to an epimeric mixture of seven-memberedmixed-acetals, presumably through the addition of methanol tooxocarbenium ion 11B.

[0321] However, the desired sense of cyclopropane opening, under theinfluence of the ring oxygen, was achieved by subjecting compound 10B tooxidative opening with N-iodosuccinimide. (For interestingHg(II)-induced solvolyses of cyclopropanes that are conceptually similarto the conversion of 10B to 12B, see: Collum, D. B.; Still, W. C.;Mohamadi, F. J. Amer. Chem. Soc., 1986, 108, 2094; Collum, D. B.;Mohamadi, F.; Hallock, J. S.; J. Amer. Chem. Soc., 1983, 105, 6882.Following this precedent, a Hg(II)-induced solvolysis of cyclopropane10B was achieved, although this transformation proved to be lessefficient than the reaction shown in FIG. 8.) The intermediateiodomethyl compound, obtained as a methyl glycoside 12B, when exposed tothe action of tri-n-butyltinhydride gave rise to pyran 13B containingthe geminal methyl groups. Protection of this alcohol (see 13B→14B),followed by cleavage of the glycosidic bond, revealed the acyclicdithiane derivative 15B which can serve as a functional version of thehypothetical aldehyde 3B.

[0322] Possible ways of combining fragments relating to 2B and 3B in afashion to reach epothilone and congeners thereof were examined. In viewof the studies of Schrock (Schrock, R. R., et al., J. Am. Chem. Soc.,1990, 112, 3875) and Grubbs (Schwab, P. et al., Angew. Chem. Int. Ed.Engl., 1995, 34, 2039; Grubbs, R. H.; Miller, S. J. Fu, G. C. Acc. Chem.Res., 1995, 28, 446; Schmalz, H.-G., Angew. Chem. Int. Ed. Engl., 1995,34, 1833) and the disclosure of Hoveyda (Houri, A. F., et al., J. Am.Chem. Soc., 1995, 117, 2943), wherein a complex lactam was constructedin a key intramolecular olefin macrocyclization step through amolybdenum mediated intramolecuar olefin in metathesis reaction(Schrock, supra; Schwab, supra), the possibility of realizing such anapproach was considered. (For other examples of ring-closing metathesis,see: Martin, S. F.; Chen, H.- J.; Courtney, A. K.; Lia, Y.; Pätzel, M.;Ramser, M N.; Wagman, A. S. Tetrahedron, 1996, 52, 7251; Furstner, A.;Langemann, K. J. Org. Chem., 1996, 61, 3942.) The matter was firstexamined with two model Ω-unsaturated acids 16B and 17B which were usedto acylate alcohol 2B to provide esters 18B and 19B, respectively (seeFIG. 9). These compounds did indeed undergo olefin metathesismacrocyclization in the desired manner under the conditions shown. Inthe case of substrate 18B, compound set 20B was obtained as a mixture ofE- and Z-stereoisomers [ca. 1:1]. Diimide reduction of 20B was thenconducted to provide homogeneous 22B. The olefin methathesis reactionwas also extended to compound 19B bearing geminal methyl groupscorresponding to their placement at C4 of epothilone A. Olefinmetathesis occurred, this time curiously producing olefin 21B as asingle entity in 70% yield (stereochemisty tentatively assigned as Z.)Substantially identical results were obtained through the use ofSchrock's molybdenum alkylidene metathesis catalyst.

[0323] As described above, olefin metathesis is therefore amenable tothe challenge of constructing the sixteen membered ring containing boththe required epoxy and thiazolyl functions of the target system. It ispointed out that no successful olefin metathesis reaction has yet beenrealized from seco-systems bearing a full compliment of functionalityrequired to reach epothilone. These negative outcomes may merely reflecta failure to identify a suitable functional group constraint patternappropriate for macrocylization.

[0324] The Total Synthesis of Epothilone B: Extension of the SuzukiCoupling Method

[0325] The present invention provides the first total synthesis ofepothilone A (1). D. Meng, et al., J. Org. Chem, 1996, 61, 7998 P.Bertinato, et al., J. Org. Chem, 1996, 61, 8000. A. Balog, et al.,Angew. Chem. Int. Ed. Engl., 1996, 35, 2801. D. Meng, et al., J. Amer.Chem. Soc., 1997, 119, 10073. (For a subsequent total synthesis ofepothilone A, see:

[0326] Z. Yang, et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 166.)This synthesis proceeds through the Z-desoxy compound (23) whichunderwent highly stereoselective epoxidation with 2,2-dimethyldioxiraneunder carefully defined conditions to yield the desired β-epoxide. Thesame myxobacterium of the genus Sorangium which produces 23 alsoproduces epothilone B (2). The latter is a more potent agent than 23,both in antifungal screens and in cytotoxicity/cell nucleusdisintegration assays. G. Höfle, et al., Angew. Chem. Int. Ed. Engl.1996, 35, 1567; D. M. Bollag, et al., Cancer Res. 1995, 55, 2325.

[0327] An initial goal structure was desoxyepothilone B (2C) or asuitable derivative thereof. Access to such a compound would enable thestudy of the regio- and stereoselectivity issues associated withepoxidation of the C12-C13 double bond. A key issue was the matter ofsynthesizing Z-tri-substituted olefinic precursors of 2C with highmargins of stereoselection. A synthetic route to the disubstitutedsystem (A. Balog, et al., Agnew. Chem. Int. Ed. Engl., 1996, 35, 2801)employed a palladium-mediated B-alkyl Suzuki coupling (N. Miyaura, etal., J. Am. Chem. Soc. 1989, 111, 314. (For a review, see: N. Miyaura,A. Suzuki, Chem. Rev. 1995, 95, 2457) of the Z-vinyl iodide 19 (FIG.4(A)) with borane 7C derived from hydroboration of compound 11 (FIG. 1(A)) with 9-BBN (FIG. 4(B)).)

[0328] A preliminary approach was to apply the same line of thinking toreach a Z-tri-substituted olefin (FIG. 17) en route to 2C. Two issueshad to be addressed. First, it would be necessary to devise a method toprepare vinyl iodide 8C, the tri-substituted analog of 19. If this goalcould be accomplished, a question remained as to the feasibility ofconducting the required B-alkyl Suzuki coupling reaction to reach aZ-tri-substituted olefin. The realization of such a transformation witha “B-alkyl” (as opposed to a “B-alkenyl” system) at the inter-molecularlevel, and where the vinyl iodide is not of the β-iodoenoate (orβ-iodoenone) genre, was not precedented. (For some close analogies whichdiffer in important details from the work shown here, see: N. Miyaura,et al., Bull. Chem. Soc. Jpn. 1982, 55, 2221; M. Ohba, et al.,Tetrahedron Lett., 1995, 36, 6101; C. R. Johnson, M. P. Braun, J. Am.Chem. Soc. 1993, 115, 11014.)

[0329] The synthesis of compound 8C is presented in FIG. 16 . The routestarted with olefin 10C which was prepared by catalytic asymmetricallylation of 9C (G. E. Keck, et al., J. Am. Chem. Soc., 1993, 115,8467) followed by acetylation. Site-selective dihydroxylation of 10Cfollowed by cleavage of the glycol generated the unstable aldehyde 11C.Surprisingly, the latter reacted with phosphorane 12C (J. Chen, et al.,Tetrahedron Lett., 1994, 35, 2827) to afford the Z-iodide 8C albeit inmodest overall yield. Borane 7C was generated from 11 as describedherein. The coupling of compound 7C and iodide 8C (FIG. 16) could beconducted to produce the pure Z-olefin 13C.

[0330] With compound 13C in hand, protocols similar to those employed inconnection with the synthesis of 23 could be used. (A. Balog, et al.,Angew. Chem. Int. Ed. Engl., 1996, 35, 2801). Thus, cleavage of theacetal linkage led to aldehyde 14C which was now subjected tomacroaldolization (FIG. 17). The highest yields were obtained bycarrying out the reaction under conditions which apparently equilibratethe C3 hydroxyl group. The 3R isomer was converted to the required 3Sepimer via reduction of its derived C3-ketone (see compound 15C). Thekinetically controlled aldol condensation leading to the natural 3Sconfiguration as discribed in the epothilone A series was accomplished.However, the overall yield for reaching the 3S epimer is better usingthis protocol. Cleavage of the C-5 triphenyisilyl ether was followedsequentially by monoprotection (t-butyldimethylsilyl) of the C3hydroxyl, oxidation at C5 (see compound 16C), and, finally, cleavage ofthe silyl protecting groups to expose the C3 and C7 alcohols (seecompound 2C).

[0331] It was found that Z-desoxyepothilone B (2C) undergoes very rapidand substantially regio- and stereoselective epoxidation under theconditions indicated (although precise comparisons are not available,the epoxidation of 2C appears to be more rapid and regioselective thanis the case with 23) (A. Balog, et al., Angew. Chem. Int. Ed. Engl.,1996, 35, 2801), to afford epothilone B (2) identical with an authenticsample (¹H NMR, mass spec, IR, [α]_(D)). Accordingly, the presentinvention dislcoses the first total synthesis of epothilone B. Importantpreparative features of the present method include the enantioselectivesynthesis of the trisubstituted vinyl iodide 8C, the palladium-mediatedstereospecific coupling of compounds 7C and 8C to produce compound 13C(a virtually unprecedented reaction in this form), and the amenabilityof Z-desoxyepothilone B (2C) to undergo regio- and stereoselectiveepoxidation under appropriate conditions.

[0332] Desmethylepothilone A

[0333] Total syntheses of epothilones A and B have not been previouslydisclosed. Balog, A., et al., Angew. Chem., Int. Ed. Engl. 1996, 35,2801; Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engl. 1997, 36,166. Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engl. 1997, 36,525; Schinzer, D., et al., Angew. Chem., Int. Ed. Engl. 1997, 36, 523.Su, D. -S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757. The modeof antitumor action of the epothilones closely mimics that of Taxol™.Höfle, G., et al., H. Angew. Chem., Int. Ed. Engl. 1996, 35, 1567.Although Taxol™ (paclitaxel) is a clinically proven drug, itsformulation continues to be difficult. In addition, taxol induces themultidrug resistance (MDR) phenotype. Hence, any novel agent that hasthe same mechanism of action as taxol and has the prospect of havingsuperior therapeutic activity warrants serious study. Bollag, D. M., etal., Cancer Res. 1995, 55, 2325.

[0334] The present invention provides epothilone analogs that are moreeffective and more readily synthesized than epothilone A or B. Thesyntheses of the natural products provide ample material for preliminarybiological evaluation, but not for producing adequate amounts for fulldevelopment. One particular area where a structural change could bringsignificant relief from the complexities of the synthesis would be inthe deletion of the C8 methyl group from the polypropionate domain (seetarget system 3D). The need to deal with this C8 chiral centercomplicates all of the syntheses of epothilone disclosed thus far.Deletion of the C8 methyl group prompts a major change in syntheticstrategy related to an earlier diene-aldehyde cyclocondensation route.Danishefsky, S. J. Chemtracts 1989, 2, 273; Meng, D., et al., J. Org.Chem. 1996, 61, 7998; Bertinato, P., et al., J. Org. Chem. 1996, 61,8000.

[0335] As shown in FIG. 20, asymmetric crotylation (87% ee) of 4D(Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919), followedby protection led to TBS ether 5D. The double bond was readily cleavedto give aldehyde 6D. The aldehyde was coupled to the dianion derivedfrom t-butyl isobutyrylacetate to provide 7D. The ratio of the C_(5S)(7D): C_(5R) compound (not shown) is ca 10:1. That the Weiler-typeβ-ketoester dianion chemistry (Weiler, L. J. Am. Chem. Soc. 1970, 92,6702.; Weiler, L.; Huckin, S. N. J. Am. Chem. Soc. 1974, 96, 1082) canbe conducted in the context of the isobutyryl group suggested severalalternate approaches for still more concise syntheses. Directedreduction of the C₃ ketone of 7D following literature precedents (Evans,D. A., et al., J. Org. Chem. 1991, 56, 741), followed by selectivesilylation of the C₃ hydroxyl gave a 50% yield of a 10:1 ratio of therequired C_(3S) (see compound 8D) to C_(3R) isomer (not shown).Reduction with sodium borohydride afforded a ca. 1:1 mixture of C₃epimers. The carbinol, produced upon debenzylation, was oxidized to analdehyde which, following methylenation through a simple Wittigreaction, afforded olefin 9D. Treatment of this compound with TBSOTfprovided ester 10D which was used directly in the Suzuki coupling withthe vinyl iodide 12D.

[0336] The hydroboration of 10D with 9-BBN produced intermediate 11Dwhich, on coupling with the vinyl iodide 12D and in situ cleavage of theTBS ester led to 13D (FIG. 21). After de-acetylation, the hydroxy acid14D was in hand. Macrolactonization of this compound (Boden, E. P.;Keck, G. E. J. Org. Chem. 1985, 50, 2394) produced 15D which, afterdesilylation, afforded C₈-desmethyldesoxyepothilone (16D). Finally,epoxidation of this compound with dimethyldioxirane produced the goalstructure 3D. The stereoselectivity of epoxidation was surprisingly poor(1.5:1) given that epoxidation of desoxyepothilone A occurred with >20:1stereoselectivity. Deletion of the C₈ methyl group appears to shift theconformational distribution of 16D to forms in which the epoxidation bydimethyl dioxirane is less β-selective. It is undetermined whether theeffect of the C₈ methyl on the stereoselectivity of epoxidation bydimethydioxirane and the dramatic reduction of biological activity arerelated.

[0337] Compounds 3D and 16D were tested for cytotoxicity in cellcultures and assembly of tubulin in the absence of GTP. Microtubuleprotein (MTP) was purified from calf brains by two cycles of temperaturedependent assembly and disassembly. Weisenberg, R. C. Science 1972, 177,1104. In control assembly experiments, MTP (1 mg/mL) was diluted inassembly buffer containing 0.1 M MES (2-(N-morpholino) ethanesulfonicacid), 1 mM EGTA, 0.5 mM MgCl₂, 1 mM GTP and 3M glycerol, pH 6.6. Theconcentration of tubulin in MTP was estimated to be about 85%. Assemblywas monitored spectrophotometrically at 350 nm, 35° C. for 40 min byfollowing changes in turbidity as a measure of polymer mass. Gaskin, F.;Cantor, C. R.; Shelanksi, M. L. J. Mol. Biol. 1974, 89, 737. Drugs weretested at a concentration of 10 μM, in the absence of GTP. Microtubuleformation was verified by electron microscopy. To determine thestability of microtubules assembled in the presence of GTP or drug,turbidity was followed for 40 min after the reaction temperature wasshifted to 4° C.

[0338] Cytotoxicity studies showed drastically reduced activity in the8-desmethyl series. Compounds 3D and 16D were approximately 200 timesless active than their corresponding epothilone A counterparts (seeTable 1). Recalling earlier SAR findings at both C₃ and C₅, inconjunction with the findings disclosed herein, the polypropionatesector of the epothilones emerges as a particularly sensitive locus ofbiological function. Su, D.-S., et al., Angew. Chem. Int. Ed. Engl 1997,36, 757; Meng, D., et al., J. Am. Chem. Soc. 1997, 119. TABLE 1 Relativeefficacy of epothilone compounds against drug-sensitive and resistanthuman leukemic CCRF-CEM cell lines.^(a) CCRF-CEM CCRF-CEM/VBLCCRF-CEM/VM₁ Compound IC₅₀ (μM)^(b) IC₅₀ (μM)^(b) IC₅₀ (μM)^(b) 16D 5.005.75 6.29 3D 0.439 2.47 0.764 epothilone A 0.003 0.020 0.003desoxyepothilone A 0.022 0.012 0.013 epothilone B 0.0004 0.003 0.002desoxyepothilone B 0.009 0.017 0.014 paclitaxel 0.002 3.390 0.002

[0339] Biological Results

[0340] In the tables which follow, model system I is desoxyepothilone.Model system 2 has the structure:

[0341] wherein R′ and R″ are H.

[0342] Model system 3 has the structure:

TABLE 2 Relative Efficacy of Epothilone Compound Against Human LeukemicCCRF-CEM Cell Growth and Against CCRF-CEM MDR Sublines Resistant toTaxol or Etoposide IC₅₀ in μM COMPOUND CCRF-CEM CCRF-CEM/VLBCCRF-CEM/VM-1 EPOTHILONE A NATURAL 0.0035 0.0272 0.0034 EPOTHILONE ASYNTHETIC 0.0029 0.0203 0.0034 MODEL SYSTEM I [3] 271.7 22.38 11.59TRIOL ANALOG [2] 14.23 6.28 43.93 DESOXY EPOTHILONE [1] 0.002 0.0120.013 TAXOL 0.0023 2.63 0.0030 VINBLASTINE 0.00068 0.4652 0.00068 VP-16(ETOPOSIDE) 0.2209 7.388 34.51 Relative Potency of Epothilone CompoundsAgainst Human Leukemic CCRF Sublines CCRF-CEM/VBL CCRF-CEM/VM₁ (MDR CellLine) (Topo II gene mutated cell line) CCRF-CEM (Taxol Resistant)-(1143fold) (Taxol Sensitive) (Parent Cell Line) (Vinblastine Resistant)(VP-16 resistant) IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ (μM) relative to (μM)relative to (μM) relative to COMPOUND (A) Epothilone A] (B) Epothilone A(B)/(A)] (C) Epothilone A (C)/(A)] TAXOL 0.0023 [0.72] 2.63 [109.6](1143)^(a) 0.0030 [0.88] (1.30)^(a) MODEL SYSTEM I 271.7 [84906] 22.38[932.5] (0.082)^(b) 11.59 [3409] (0.043)^(b) TRIAL ANALOG 14.23 [4447]6.28 [261.7] (0.44)^(b) 43.93 [12920] (3.09)^(a) DESOXYEPOTHILONE A0.022 [6.9] 0.012 [0.5] (0.55)^(b) 0.013 [3.82] (0.59)^(b) EPOTHILONE A0.0032 [1] 0.024 [1] (7.5)^(a) 0.0034 [1] (1.06)^(a)

[0343] TABLE 2 Relative Potency of Epothilone Compounds Against HumanLeukemic CCRF Sublines CCRF-CEM/VM₁ CCRF/CEM/VBL (Topo II gene mutated(MDR Cell Line) cell line) CCRF-CEM (Taxol Resistant)-(1143 fold) (TaxolSensitive) (Parent Cell Line) (Vinblastine Resistant) (VP-16 resistant)IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ (μM) relative to (μM) relative to (μM)relative to COMPOUND (A) Epothilone A] (B) Epithilone A (B)/(A)] (C)Epothilone A (C)/(A)] TAXOL 0.0023 [0.72] 2.63 [109.6] (1143)^(a) 0.0030[0.88] (1.30)^(a) MODEL SYSTEM 271.7 [84906] 22.38 [932.5] (0.082)^(b)11.59 [3409] (0.043)^(b) I TRIAL ANALOG 14.23 [4447] 6.28 [261.7](0.44)^(b) 43.93 [12920] (3.09)^(a) DESOXYEPO- 0.022 [6.9] 0.012 [0.5](0.55)^(b) 0.013 [3.82] (0.59)^(b) THILONE A EPOTHILONE A 0.0032 [1]0.024 [1] (7.5)^(a) 0.0034 [1] (1.06)^(a)

[0344] As shown in Table 2, CCRF-CEM is the parent cell line.CCRF-CEM/VBL (MDR cell line) is 1143-fold resistant to taxol.CCRF-CEM/VM (Topo II mutated cell line) only 1.3-fold resistant totaxol.

[0345] In terms of relative potency, synthetic Epothilone is roughly thesame as natural Epothilone A. For CCRF-CEM cells, the ordering is:

[0346] Taxol≈Epothilone A>Desoxy Epothilone A>>Triol Analog>>ModelSystem I

[0347] For CCRF-CEM/VBL, the relative potency ordering is:

[0348] Desoxy Epothilone A≧Epothilone A>>Taxol >Triol Analog >ModelSystem I

[0349] For CCRF-CEM/VM, the relative potency ordering is:

[0350] Taxol Epothilone A>Desoxy Epothilone A>>Model System I>TriolAnalog

[0351] It is concluded that CCRF-CEM/VM cells are collaterally sensitiveto certain epothilone compounds. TABLE 3 Relative Efficacy of EpothiloneCompounds Against The DC-3F Hamster Lung Cell Growth and Against DC-3FMDR Sublines Resistant Actinomylin D IC₅₀ in μM COMPOUNDS DC-3FDC-3F/ADII DC-3F/ADX EPOTHILONE A NATURAL 0.00368 0.01241 0.0533EPOTHILONE A SYNTHETIC 0.00354 0.0132 0.070 MODEL SYSTEM I [3] 9.523.004 0.972 TRIOL ANALOG [2] 10.32 4.60 4.814 DESOXY EPOTHILONE [1]0.01061 0.0198 0.042 TAXOL 0.09469 3.205 31.98 VINBLASTINE 0.002650.0789 1.074 VP-16 (Etoposide) 0.03386 0.632 12.06 ACTINOMYCIN-D0.000058 0.0082 0.486 (0.05816 nm)

[0352] Concerning Table 3, experiments were carried out using the celllines DC-3F (parent hamster lung cells), DC-3F/ADII (moderatemultidrug-resistant (MDR) cells) and DC-3F/ADX (very strong MDR cells).

[0353] The relative potency of the compounds are as follows: DC-3F:Actinomycin D > Vinblastine ≧ Epothilone A (0.0036 μM) > Desoxyepothilone > VP-16 > Taxol (0.09 μM) > Model system I and triol analogDC-3F/ADX: Desoxyepothilone ≧ Epothilone A (0.06 μM) > Actinomycin D >Model system I > Vinblastine > triol analog > viablastine > taxol (32.0μM) DC-3F/ADX cells (8379-fold resistant to actinomycin D) are > 338fold (ca. 8379 fold) resistant to Taxol, VP-16, Vinblastine andActinomycin D but < 20 fold resistant to epothilone compounds.

[0354] In general, these results are similar to those for CCRF-CEMcells. TABLE 4 Three Drug Combination Analysis (Based on the MutuallyExclusive Assumption - Classical Isobologram Method) Drug A: EPOTHILONEB (#8) (μM) Drug B: TAXOL (μM) Drug C: VINBLASTINE (μM) Conditions:CCRF-CEM, 3 DRUG COMBINATION, RATIO (A:B:C: 1:5:1); EPOTHILONE + TAXOL +VINBLASTINE; EXPOSURE TIME 72 HRS; XTT ASSAY. Combination Index* Valuesat: Parameters Drug ED50 ED75 ED90 ED95 Dm (IC₅₀) (μm) m r A −000611.71561 .98327 B −00109 2.14723 .98845 C −00061 1.76186 .9919 A + B1.51545 1.38631 1.27199 1.20162 −00146 2.41547 .97168 B + C 1.432431.33032 1.23834 1.18091  .00138 2.35755 .95695 A + C .74395 .68314.62734 .59204  .00045 2.0098 .96232 A + B + C 1.37365 1.32001 1.272851.24412  .00122 2.11202 .93639 VBL → microtubule depolymerization Taxol→ microtubule polymerization Epo-B → microtubule polymerizationEpothilone B and Taxol have a similar mechanism of action(polymerization) but Epothilone B synergizes VBL whereas Taxolantagonizes VBL. Taxol + VBL → Antagonism EpoB + Taxol → AntagonismEpoB + VBL → Synergism EpoB + Taxol + VBL → Antagonism

[0355] TABLE 5 Relative cytotoxicity of epothilone compounds in vitro.IC₅₀ in μM Compounds CCRF-CEM CCRF-CEM/VLB CCR$F-CEM/VM-1 CINBLASTINE**** 0.0008 0.44 0.00049 0.0006 (0.00063 0.221 (0.332 0.00039 (0.000410.0005 ± 0.00008) 0.336 ±0.063 0.00036 ±0.00004) (52.7X)^(§) (0.7X)VP-16 0.259 6.02 35.05 0.323 (0.293 9.20 (10.33 42.24 (34.39 0.296 ±0.019) 15.76 ± 2.87) 25.89 ± 4.73) (35.3X) (117.4X) TAXOL *** 0.00214.14 0.0066 #17 * 0.090 0.254 #18 1157.6 >>1 #19 0.959 >>1 #20 * 0.0300.049 #21 — — #22 * 0.098 0.146 #23 — — #24 *** 0.0078 0.053 #25 * 0.0210.077 #26 * 0.055 0.197 #27 **** 0.0010 0.0072 Epothilone A (Syn) ***0.0021 0.015 Epothilone B (Syn) **** 0.00042 0.0017

[0356] TABLE 6 Relative potency of epothilone compounds in vitro. IC₅₀in μM Compunds CCRF-CEM CCRF-CEM/VBL CCRF-CEM/VM-1 Desoxy Epo. A 1 *0.022 0.012 0.013 2 14.23 6.28 43.93 3 271.7 22.38 11.59 4 2.119 43.012.76 5 >20 35.19 98.04 Trans- A 6 0.052 0.035 0.111 7 7.36 9.82 9.65Syn-Epo.-B 8 **** 0.00082 0.0029 0.0044 Natural B 9 **** 0.00044 0.00260.0018 Desoxy Epo. B 10 *** 0.0095 0.017 0.014 Trans. Epo. B 11 * 0.0900.262 0.094 12 0.794 >5 >5 13 11.53 5.63 14.46 8-desmethyl desoxy-tpo 145.42 5.75 6.29 8-desmethyl Mix-cis Epo 15 0.96 5.95 2.55 8-desmethylβ-Epo 15 0.439 2.47 0.764 8-demethyl α-Epo 16 7.47 16.48 0.976EPOTHILONE A *** 0.0024 (0.0027 0.0211 (0.020 0.006 (0.00613 {closeoversize brace} (Natural) 0.0031 ± 0.0003) 0.0189 ± 0.001) 0.00625 ±0.0001) (7.4X) (2.3X) EPOTHILONE B **** 0.00017 0.0017 (7.0X) 0.00077(Natural) EPOTHILONE B (Synthetic) 0.00055 0.0031 (0.00213 0.0018(0.00126 EPOTHILONE B (0.00035 ± 0.00055) , ± 0.0003) (Synthetic, larger± 0.0003) , quantity synthesis) 0.0021 (6.1X) 0.0012 (3.6X) (25.9 mg)0.00033

[0357] TABLE 7 Relative cytotoxicity of epothilone compounds in vitro.IC₅₀ CEM CEM/VBL epothilone A 0.0029 0.0203 μM μM desoxyepothilone 0.0220.012 2 14.2 6.28 3 271.7 22.4 4 2.1 43.8 5 >20 35.2 6 0.052 0.035 7 7.49.8 synthetic epothilone B 0.00082 0.00293 natural epothilone B 0.000440.00263 desoxyepothilone B 0.0095 0.0169 11 0.090 0.262 12 0.794 >5 1311.53 5.63 14 5.42 5.75 15 0.439 2.47 16 7.47 16.48 17 0.090 0.254 181157.6 >>1 19 0.959 >>1 20 0.030 0.049 21 Not Available — 22 0.098 0.14623 Not Available — 24 0.0078 0.053 25 0.0212 0.077 26 0.0545 0.197 270.0010 0.0072

[0358] TABLE 8 Chemotherapeutic Effect of Epothilone B, Taxol &Vinblastine in CB-17 Scid Mice Bearing Human CCRF-CEM and CCRF-CEM/VBLXenograft¹ Average weight change Average tumor volume Tumor Drug² DoseDay 0 Day 7 Day 12 Day 17 Day 22 Day 7 Day 12 Day 17 Day 22 CCRF-CEM 024.4 +0.2 +0.4 +0.1 +0.5 1.0³ 1.00 1.00 1.00 Epo B 0.7⁴ 24.7 −0.1 −0.7−1.4 +0.3 1.0 0.53 0.48 0.46 1.0⁵ 25.0 +0.1 −1.5 −2.4 +0.1 1.0 0.46 0.350.43 Taxol 2.0 25.1 −0.1 −1.1 −1.5 −0.3 1.0 0.39 0.29 0.28 4.0 25.1 −0.2−1.7 −1.9 −0.3 1.0 0.37 0.23 0.19 VBL 0.2 25.9 +0.2 −0.8 −1.5 −0.3 1.00.45 0.25 0.29 0.4 25.0 −0.1 −1.4 −1.8 −0.7 1.0 0.31 0.27 0.30 CCRF-CEM0 26.3 −0.3 +0.1 −0.3 +0.4 1.0 1.00 1.00 1.00 /VBL Epo B 0.7 25.8 +0.1−0.7 −1.0 −0.2 1.0 0.32 0.40 0.33 1.0⁶ 26.0 −0.2 −1.3 −2.1 −0.5 1.0 0.410.27 0.31 Taxol 2.0 26.1 0 −0.9 −1.5 −0.1 1.0 0.60 0.58 0.70 4.0 26.0 0−1.4 −1.6 −0.9 1.0 0.79 0.55 0.41 VBL 0.2 25.9 −0.3 −0.8 −1.4 −0.3 1.00.86 0.66 0.67 0.4 25.9 0 −1.2 −1.8 −0.5 1.0 1.02 0.57 0.62

[0359] In summary, epothilones and taxol have similar modes of action bystabilizing polymerization of microtubules. However, epothilones andtaxol have distinct novel chemical structures.

[0360] MDR cells are 1500-fold more resistant to taxol (CCRF-CEM/VBLcells), epothilone A showed only 8-fold resistance and epothilone Bshowed only 5-fold resistance. For CCRF-CEM cells, Epo B is 6-fold morepotent than Epo A and 10-fold more potent than Taxol. Desoxyepothilone Band compd #24 are only 34-fold less potent than Taxol and compound #27is >2-fold more potent than Taxol. Finally, Taxol and vinblastine showedantagonism against CCRF-CEM tumor cells, whereas the combination of EpoB+vinblastine showed synergism.

[0361] Relative Cytotoxicity of Epothilones against Human Leukemic Cellsin Vitro is in the order as follows:

[0362] CCRF-CEM Leukemic Cells

[0363] Epo B (IC₅₀=0.00035 μM; Rel.Value=1)>VBL(0.00063;1/1.8)>#27(0.0010;1/2.9)>Taxol (0.0021; 1/6)>Epo A(0.0027; 1/7.7)>#24(0.0078; 1/22.3)>#10 (0.0095; 1/27.1)>#25 (0.021;1/60)>#1 (0.022; 1/62.8)>#20 (0.030; 1/85.7)>#6 (0.052; 1/149)>#260.055; 1/157)>#17 (0.090; 1/257)>VP-16 (0.29; 1/8.29)>#15 (0.44;1/1257)>#19 (0.96; 1/2943)

[0364] CCRF-CEM/VBL MDR Leukemic Cells

[0365] Epo B (0.0021; 1/6* [1]**)>#27 (0.0072; 1/20.6)>#1 (0.012;1/34.3)>#10 (0.017; 1/48.6)>Epo A (0.020; 1/57.1 [1/9.5])>#6 (0.035)>#20(0.049)>#24 (0.053)>#25 (0.077)>#22 (0.146)>#26 (0.197)>#17 (0.254)>#11(0.262)>VBL (0.332; 1/948.6 [1/158.1])>Taxol (4.14; 1/11828[1/1971.4])>VP-16 (10.33; 1/29514 [1/4919])

[0366] *Potency in parentheses is relative to Epo B in CCRF-CEM cells.

[0367] **Potency in square brackets is relative to Epo B in CCRF-CEM/VBLMDR cells.

[0368] As shown in Table 9, treatment of MX-1 xenograft-bearing nudemice with desoxyepothilone B (35 mg/kg, 0/10 lethality), taxol (5 mg/kg,2/10 lethality; 10 mg/kg, 2/6 lethality) and adriamycin (2 mg/kg, 1/10lethality; 3 mg/kg, 4/6 lethality) every other day, i.p. beginning day 8for 5 doses resulted in a far better therapeutic effect fordesoxyepothilone B at 35 mg/kg than for taxol at 5 mg/kg and adrimycinat 2 mg/kg with tumor volume reduction of 98%, 53% and 28%,respectively. For the desoxyepothilone B-treated group, 3 out of 10 micewere found with tumor non-detectable on day 18. (See FIG. 46)

[0369] Extented treatment with desoxyepothilone B (40 mg/kg, i.p.)beginning day 18 every other day for 5 more doses resulted in 5 out of10 mice with tumor disappearing on day 28 (or day 31). See Table 10. Bycontrast, the extended treatment with taxol at 5 mg/kg for five moredoses resulted in continued tumor growth at a moderate rate, and 2 outof 10 mice died of toxicity.

[0370] Toxicity studies with daily i.p. doses of desoxyepothilone B (25mg/kg, a very effective therapeutic dose as indicated in earlierexperiments) for 4 days to six mice resulted in no reduction in averagebody weight. (Table 13; FIG. 47) By contrast, epothilone B (0.6 mg/kg,i.p.) for 4 days to eight mice resulted in 33% reduction in average bodyweight; all eight mice died of toxicity between day 5 and day 7. TABLE 9Therapeutic Effect of Desoxyepothilone B, Taxol, and Adriamycin in NudeMice Bearing Human MX-1 Xenograft^(a) Average Body Weight Change AverageTumor Volume Tumor Dose (g) (T/C) Died Disappear- Drug (mg/kg) Day 8 1012 14 16 18 Day 10 12 14 16 18 ance Control  0 24.6 −0.1 +1.0 +1.0 +1.3+1.8 1.00 1.00 1.00 1.00 1.00 0/10 0/10 Desoxyepothilone B 35 23.0 −0.1+0.7 −0.3 −1.7 −1.6 0.42 0.28 0.07 0.04 0.02 0/10 3/10 Taxol  5 24.0−1.3 −0.8 −1.4 −1.9 −1.8 0.58 0.36 0.34 0.42 0.47 2/10 0/10 10 24.3 −1.0−1.0 −2.3 −3.5 −3.8 0.85 0.40 0.21 0.20 0.12 2/6  1/6  Adriamycin  2^(b)23.9 +0.3 0 −1.4 −1.9 −2.0 0.94 0.88 1.05 0.69 0.72 1/10 0/10  3^(c)22.4 +1.3 −0.2 −1.5 −2.1 −2.3 0.72 0.54 0.56 0.51 0.36 4/6  0/6 

[0371] TABLE 10 Extended Experiment of Desoxyepothilone B, Taxol,Cisplatin and Cyclophophamide in Nude Mice Bearing Human MX-1Xenograft^(a) Average Body Weight Change Average Tumor Dose (g) TumorDisappearance Disappearance # Drug (mg/kg) Day 8 20 22 24 26 28 Day 2022 24 26 28 Duration (Day) Died Desoxyepo B 40 23.0 −1.7 −2.4 −2.4 −1.4−1.2 2/10^(b) 2/10 3/10 5/10 5/10 44 (5/10) 0/10 Taxol 5 24.0 −1.6 −0.3+0.1 −0.6 −0.4 0/10  0/10 0/10 0/10 0/10 2/10 10 No extended test 1/6 onday16 Reappear on day 38 2/6 

[0372] TABLE 11 Toxicity of Epothilone B and Desoxyepothilone B innormal nude mice. Dose and Schedule Group (mg/kg) Number of mice DiedDisappearance Duration Control 4 0 Epothilone B^(a) 0.6 QD × 4 8 8Desoxyepothilone B  25 QD × 4 6 0

[0373] TABLE 12 Therapeutic Effect of Epothilone B, Desoxyepothilone Band Taxol in B6D2F, Mice Bearing B16 Melanoma^(a) Average Weight ChangeAverage Tumor Volume # Dose (g) (T/C) Mice Drug (mg/kg) Day 0 3 5 7 9 11Day 5 7 9 11 Died Control 0 26.5 −0.2 0 −0.2 0 +1.0 1.00 1.00 1.00 1.000/15 Epothilone B 0.4 QDx6^(b) 27.1 −0.2 −0.6 −1.1 −3.4 −3.9 1.08 1.071.27 1.07 1/8  0.8 QDx5^(c) 27.0 0 −0.8 −3.1 −4.7 −4.7 0.57 0.89 0.460.21 5/8  Desoxyepothilone B 10 QDx8 27.0 −0.7 −0.9 −1.1 −1.5 −0.3 0.230.22 0.51 0.28 0/6  20 QD1-4,7-8 26.9 −1.3 −2.2 −1.3 −1.6 −0.8 0.59 0.630.58 0.33 0/6  Taxol 4 QDx8 26.7 +0.1 +0.2 +0.3 +0.4 +0.8 0.62 0.39 0.560.51 0/8  6.5 QDx8 26.7 +0.1 +0.3 +0.3 +0.4 +1.7 0.19 0.43 0.20 0.540/8 

[0374] TABLE 13 Therapeutic Effect of Desoxyepothilone B, Epothilone B,Taxol and Vinblastine in Nude Mice Bearing Human MX-1 Xenograft^(a).Average Body Weight Change Average Tumor Colume Dose (g) (T/C) Drug(mg/kg) Day 7 11 13 15 17 Day 11 13 15 17 Note Control 27.9 +0.8 +1.1+1.9 +0.6 1.00 1.00 1.00 1.00 0/8 died Desoxyepothilone B 15 27.1 +0.8+1.1 +1.6 +1.5 0.65 0.46 0.49 0.41 0/6 died 25^(b) 27.0 +0.4 +0.7 +1.0+0.7 0.38 0.11 0.05 0.04 0/6 died (1/6 cured on day 35) Epothilon B 0.326.9 +0.5 +0.4 −0.3 −1.2 1.00 0.71 0.71 0.84 0/7 died 0.6^(c) 27.4 −0.3−1.3 −2.1 −2.1 1.08 0.73 0.81 0.74 3/7 died Taxol 5 26.9 −0.1 +0.4 +1.1+1.2 0.54 0.46 0.40 0.45 0/7 died 10^(d) 27.6 −2.7 −1.1 −0.3 +2.2 0.430.37 0.12 0.11 4/7 died Vinblastine 0.2 25.7 +0.6 +1.4 +2.3 +2.9 0.650.54 0.56 0.88 0/7 died 0.4^(c) 26.4 +0.8 +0.5 +1.9 +2.1 0.80 0.56 0.830.88 1/7 died

[0375] TABLE 14 Toxicity of Hematology and Chemistry of DesoxyepothiloneB, and Taxol in Nude Mice Bearing Human MX-1 Xenograft^(a)Hematology^(b) WBC Chemistry^(b) Dose Total Neutrophils Lymph RBC PLTGOT GPT Drug (mg/kg ip) (10³/mm³) (%) (%) (10³/mm³) (10⁶/mm³) (U/L)(U/L) Control 12.9 38 61 8.1  800 (n = 4) 203 45 (n '2 4) Desoxyepo- 25and 35^(c) 11.8 48 48 8.4  700 (n = 6) 296 55 (n '2 3) thilone B Taxol 5and 6^(d) 10.9 51 48 6.1 1083 (n = 5) 438 79 (n '2 5) Normal range^(c)6.91˜12.9 8.25˜40.8 62˜90 10.2˜ 190˜340 260 138.7 12.0

[0376] TABLE 15 Therapeutic Effect of Desoxyepothilone B, Taxol,Adriamycin, and Camptothecin in Nude Mice Bearing MDR Human MCF-7/AdrTumor. Average Body Weight Change Average Tumor Volume Dose (g) (T/C)Drug (mg/kg) Day 8 11 13 15 17 Day 11 13 15 17 Died Control 0 25.0 +2.0+2.6 +3.1 +3.7 1.00 1.00 1.00 1.00 0/8 DesoxyEpoB 35 25.0 +0.3 +0.7 +0.6+0.8 0.31 0.27 0.30 0.34 0/8 Taxol 6 25.3 +1.7 +1.8 +0.8 +0.9 0.57 0.660.85 0.90 0/8 12 24.5 +0.7 −1.3 −2.4 0 0.50 0.51 0.32 0.40 3/6Adriamycin 1.8 25.6 +0.2 −0.4 −0.6 −0.4 0.70 0.68 0.84 0.78 0/8 3 24.6+0.5 −1.5 −3.2 −1.6 0.66 0.83 0.57 0.53 3/6 Camptothecin 1.5 24.4 +1.1+0.9 +1.7 +1.4 1.08 0.72 0.61 0.72 0/8 3.0 24.5 −0.6 −0.4 −0.8 −0.9 0.950.76 0.61 0.43 0/6

[0377] As evident from Table 15, desoxyepothilone B performssignificantly better than taxol, vinblastine, adriamycin andcamptothecin against MDR tumor xenografts (human mammary adeoncarcinomaMCF-7/Adr xenografts). This drug-resistant tumor grows very aggressivelyand is refractory to taxol and adriamycin at half their lethal doses.Taxol at 6 mg/kg i.p. Q2Dx5 reduced tumor size only 10% while adriamycinresulted in only a 22% reduction on day 17. Whereas, desoxyepothilone Bat 35 mg/kg reduced tumor size by 66% on day 17 and yet showed noreduction in body weight or apparent toxicity. Even at the LD₅₀ dosagefor taxol (1 2 mg/kg) or adriamycin (3 mg/kg), desoxyepothilone B stillperformed more effectively. By comparison, camptothecin at 1.5 and 3.0mg/kg reduced tumor size by 28% and 57%, respectively. Overall, incomparison with the four important anticancer drugs in current use,i.e., taxol, adriamycin, vinblastine and camptothecin, desoxyepothiloneB showed superior chemotherapeutic effect against MDR xenografts. TABLE16 Extended Experiment of Desoxyepothilone B, Taxol in Nude Mice BearingHuman MX-1 Xenograft^(a) Average Tumor Average Body Weight Change TumorDisappear Dose (g) Disappearance Duration Drug (mg/kg) Day 8 20 22 24 2628 Day 20 22 24 26 28 (Day) Died Desoxyepo B 40 23.0 −1.7 −2.4 −2.4 −1.4−1.2 2.10^(b) 2/10 3/10 5/10 5/10 44_((5/10)) 0/10 Taxol 5 24.0 −1.6−0.3 +0.1 −0.6 −0.4 0/10 0/10 0/10 0/10 0/10 2/10 10 No Extended Test1/6 on day 16, Reappear 2/6_((0/6)) on day 38

[0378] As evident from Table 16, extended treatment of nude mice bearinghuman MX-1 xenografts with desoxyepothilone B results in complete tumordisappearance, with no mortality in any test animals. In conclusion,treatment with desoxyepothilone B shows remarkable specificity withrespect to tumor toxicity, but very low normal cell toxicity. TABLE 17Therapeutic Effects of Desoxyepothilone B, Taxol in Nude Mice BearingMX-1 Xenograft. # Died of

NTROL Treatment Schedule toxicity

8 10 12 14 16 18 20 0/10

nor Size 19 78 151 372 739 1257 1991 Sacrificed (n = 10)

n³) ±2 ±8 ±15 ±55 ±123 ±184 ±331

OXYEPOTHILONE B

se

edule 35 mg/kg on day 40 mg/kg on day No Treatment

8 10 12 14 16 18 20 22 24 26 28 30 45 47 50  60 

nor Size

use 1 15 15 40 40 15 32 30 30 30 30 0 0 0 24  S* —

use 2 23 23 15 15 15 15 30 48 48 0 30 48 900 1200 S —

use 3 15 60 90 105 105 126 96 150 180 0 48 64 600 600 S —

use 4 21 38 38 0 0 10 8 8 8 8 0 0 0 0 0 0

use 5 12 23 50 12 0 4 0 0 0 0 0 0 0 0 0 0

use 6 15 40 32 8 8 8 8 12 12 12 12 30 120 120 S —

use 7 21 30 15 15 8 8 8 8 8 8 8 8 180 280 S —

use 8 20 48 70 15 15 8 8 0 0 0 0 0 0 8 S —

use 9 25 50 40 15 8 0 0 0 0 0 0 0 0 0 4 4

use 10 20 38 38 38 38 25 25 25 0 0 15 15 100 100 S —

OL 2/10

se

edule 5 mg/kg on day 5 mg/kg on day

8 10 12 14 16 18 20 22 24 26 28 30 45 47 50 60

nor Size 17 45 54 128 311 596 1114 1930 2285 S (n = 10)

±2 ±7 ±13 ±42 ±115 ±151 ±346 ±569 ±597 Extended studies → Extendedobservations → Experiment ended

[0379] TABLE 18 Toxicity of Epothilone B and Desoxyepothilone B innormal nude mice Dose and Schedule Group (mg/kg) Number of mice DiedControl 4 0 Epothilone B^(a) 0.6 QD × 4 8 8 Desoxyepothilone B  25 QD ×4 6 0

What is claimed is:
 1. A compound having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CHY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; and wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N-NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or
 3. 2. Thecompound of claim 1 having the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,

(CH₂)₃—OH.
 3. A compound having structure:

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CHY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; and wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N-NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or
 3. 4. Thecompound of claim 3 having the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl or n-hexyl.
 5. Acompound having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁, and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is -CHY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; and wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N-NR₄R₅, wherein R₃, R₄ and R₅, are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or
 3. 6. Thecompound of claim 5 having the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl orhydroxypropyl.
 7. A compound having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁, and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CHY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; and wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅, are independently H or alinear or branched chain alkyl or alkoxy; and wherein n is 0, 1, 2, or3.
 8. A compound having the structure:


9. A compound having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR*)₂, (SR*)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n),—S)—; wherein R* is a linear or branched alkyl,substituted or unsubstituted aryl or benzyl; wherein R₂B is a linear,branched or cyclic alkyl or substituted or unsubstituted aryl or benzylboranyl moiety; and wherein n is 2, 3 or
 4. 10. A compound having thestructure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR*)₂, (SR*)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n),—S)—; wherein R* is a linear or branched alkyl,substituted or unsubstituted aryl or benzyl; wherein R₂B is a linear,branched or cyclic alkyl or substituted or unsubstituted aryl or benzylboranyl moiety; wherein Y is OH, linear or branched chain alkoxy,trimethylsilyloxy, t-butyldimethylsilyloxy or methyldiphenysilyloxy; andwherein n is 2, 3 or
 4. 11. A compound having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiaryisilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)—;and wherein n is 2, 3 or
 4. 12. The compound of claim 11 wherein R′ isTBS, R″ is TPS and X is (OMe)₂.
 13. A compound having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is a halogen;wherein R″ is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; and wherein Y is H or linear or branched chain alkyl.;wherein R′ is H, linear or branched chain alkyl, hydroxymethyl,hydroxypropyl, alkyl carboxaldehyde, alkyl carboxaldehyde linear orcyclic acetal; and X is a halide.
 14. The compound of claim 13 wherein Ris acetyl and X is iodo.
 15. A compound having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)—;and wherein n is 2, 3 or
 4. 16. A compound having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is a halogen;wherein R′ is H, linear or branched chain alkyl, alkyl carboxaldehyde,alkyl carboxaldehyde linear or cyclic acetal; wherein R″ is H, linear orbranched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl,3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl,2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and wherein Y is H orlinear or branched chain alkyl.
 17. A compound having the structure:

wherein R is hydrogen, methyl, ethyl, n-propyl, n-hexyl, CO₂Et,

CH₂OH; or (CH₂)₃—OH; wherein R′ and R″ are independently hydrogen, alinear or branched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; and wherein Z ishydrogen, or linear or branched chain alkyl.
 18. A method of preparing aZ-haloalkene ester having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein R′ is hydrogen,methyl, ethyl, n-propyl, n-hexyl, CO₂Et,

CH₂OH or (CH₂)₃—OH; and wherein X is a halogen, which comprises (a)oxidatively cleaving a compound having the structure:

 under suitable conditions to form an aldehyde intermediate; and (b)condensing the aldehyde intermediate with a halomethylene transfer agentunder suitable conditions to form the Z-haloalkene ester.
 19. The methodof claim 18 wherein X is iodine.
 20. The method of claim 18 wherein thehalomethylene transfer agent is Ph₃P═CR′I or (Ph₃P⁺CHR′I)I⁻.
 21. Amethod of preparing an optically pure compound having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)condensing an allylic organometallic reagent with an unsaturatedaldehyde having the structure:

 under suitable conditions to form an alcohol, and, optionallyconcurrently therewith, optically resolving the alcohol to form anoptically pure alcohol having the structure:

(b) alkylating or acylating the optically pure alcohol formed in step(a) under suitable conditions to form the optically pure compound. 22.The method of claim 21 wherein the allylic organometallic reagent is anallyl(trialkyl)stannane.
 23. The method of claim 21 wherein thecondensing step is effected using a reagent comprising a titaniumtetraalkoxide and an optically active catalyst.
 24. The method of claim23 wherein the optically active catalyst is S(−)BINOL.
 25. A method ofpreparing an open-chain aldehyde having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)cross-coupling a haloolefin having the structure:

 wherein R is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsily, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, and X is a halogen, with a terminalolefin having the structure:

 wherein (OR′″)₂ is (OR₀)₂, (SR₀)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)—or —(S—(CH₂)_(n)—S)— where R₀ is a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl; and wherein n is 2, 3 or 4, undersuitable conditions to form a cross-coupled compound having thestructure:

 wherein Y is CH(OR*)₂ where R* is a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryloxyalkyl; and (b)deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain compound.
 26. A method ofpreparing an epothilone having the structure:

which comprises: (a) deprotecting a cyclized compound having thestructure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, under suitable conditionsto form a deprotected cyclized compound and oxidizing the deprotectedcyclized compound under suitable conditions to form a desoxyepothilonehaving is the structure:

 and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone.
 27. A method of preparing anepothilone precursor having the structure:

wherein R₁ is hydrogen or methyl; wherein X is O, or a hydrogen and OR″,each singly bonded to carbon; and wherein R₀, R′ and R″ areindependently hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises (a) coupling a compoundhaving the structure:

wherein R is an acetyl, with an aldehyde having the structure:

wherein Y is oxygen, under suitable conditions to form an aldolintermediate and optionally protecting the aldol intermediate undersuitable conditions to form an acyclic epthilone precursor having thestructure:

(b) subjecting the acylic epothilone precursor to conditions leading tointramolecular olefin metathesis to form the epothilone precursor. 28.The method of claim 27 wherein the conditions leading to intramolecularolefin metathesis require the presence of an organometallic catalyst.29. The method of claim 27 wherein the catalyst is a Ru or Mo complex.30. A pharmaceutical composition for treating cancer comprising acompound of claim 1, 3, 5, 7, or 8 and a pharmaceutically suitablecarrier.
 31. A method of treating cancer in a subject sufferingtherefrom comprising administering to the subject a therapeuticallyeffective amount of a compound of claim 1, 3, 5, 7 or 8 and apharmaceutically suitable carrier.
 32. The method of claim 31 whereinthe cancer is a solid tumor.
 33. The method of claim 31 wherein thecancer is breast cancer.
 34. A method of preparing a Z-iodoalkene esterhaving the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises (a)coupling a compound having the structure:

with a methyl ketone having the structure:

wherein R′ and R″ are independently a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryl or benzyl, under suitableconditions to form a compound having the structure:

(b) treating the compound formed in step (a) under suitable conditionsto form a Z-iodoalkene having the structure:

and (c) deprotecting and acylating the Z-iodoalkene formed in step (b)under suitable conditions to form the Z-iodoalkene ester.
 35. A methodof preparing an open-chain aldehyde having the structure:

wherein R is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R′ and R″ are independentlyhydrogen, a linear or branched alkyl, substituted or unsubstituted arylor benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, whichcomprises: (a) cross-coupling a haloolefin having the structure:

wherein X is a halogen, with a terminal hydroborane having thestructure:

wherein R*₂B is a linear, branched or cyclic alkyl or substituted orunsubstituted aryl or benzyl boranyl moiety; wherein Y is (OR₀)₂,(SR₀)₂,—(O—(CH₂) _(n)—O)—, —(O—(CH₂)_(n)—S)—or —(S—(CH₂)_(n)—S)— whereR₀ is a linear or branched alkyl, substituted or unsubstituted aryl orbenzyl; and wherein n is 2, 3 or 4, under suitable conditions to form across-coupled compound having the structure:

(b) deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain aldehyde.
 36. The method ofclaim 35 wherein R is acetyl; R′ is TBS; R″ is TPS; R*₂B is derived from9-BBN; and Y is (OMe)₂.
 37. A method of preparing a protected epothilonehaving the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkyl-arylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)monoprotecting a cyclic diol having the structure:

under suitable conditions to form a cyclic alcohol having the structure:

and (b) oxidizing the cyclic alcohol formed in step (a) under suitableconditions to form the protected epothilone.
 38. The method of claim 37wherein R′ and R″ are TBS.
 39. A method of preparing an epothilonehaving the structure:

which comprises: (a) deprotecting a protected cyclic ketone having thestructure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, under suitable conditionsto form a desoxyepothilone having the structure:

and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone.
 40. The method of claim 39wherein R′ and R″ are TBS.
 41. A method of preparing a cyclic diolhaving the structure:

wherein R′ is a hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises: (a) cyclizing anopen-chain aldehyde having the structure:

wherein R′ is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R″ is a hydrogen, a linearor branched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl under suitableconditions to form an enantiomeric mixture of a protected cyclic alcoholhaving the structure:

said mixture comprising an α- and a β-alcohol component; (b) optionallyisolating and oxidizing the α-alcohol formed in step (a) under suitableconditions to form a ketone and thereafter reducing the ketone undersuitable conditions to form an enantiomeric mixture of the protectedcyclic alcohol comprising substantially the β-alcohol; and (c) treatingthe protected cyclic alcohol formed in step (a) or (b) with adeprotecting agent under suitable conditions to form the cyclic diol.42. The method of claim 41 wherein R′ is TBS and R″ is TPS.
 43. Apurified compound having the structure:

wherein R is hydrogen, methyl, ethyl, propyl, hexyl, hydroxymethyl orhydroxypropyl; wherein X is O; and wherein R₀, R′ and R″ areindependently hydrogen or acetyl.
 44. A purified compound having thestructure:

wherein R₁ is hydrogen, methyl, ethyl, propyl, hexyl, hydroxymethyl orhydroxypropyl; wherein X is O; and wherein R₀, R′ and R″ areindependently hydrogen or acetyl.
 45. A composition comprising an amountof the compound of claim 1, 2, 3, 4, 5, 6, 7, 8, 43 or 44 effective toinhibit the growth of multidrug resistant cells and a pharmaceuticallyacceptable carrier.
 46. The composition of claim 45, further comprisingan amount of a cytotoxic agent.
 47. The composition of claim 46, whereinthe cytotoxic agent is an anticancer agent.
 48. The composition of claim47, wherein the anticancer agent is adriamycin.
 49. The composition ofclaim 47, wherein the anticancer agent is vinblastin.
 50. Thecomposition of claim 47, wherein the anticancer agent is paclitaxel. 51.The composition of claim 45, wherein the effective amount of thecompound is between about 0.01 mg/kg to about 25 mg/kg of body weight.52. A method of inhibiting the growth of multidrug resistant cellscomprising contacting the multidrug resistant cells with an amount ofthe compound of claim 1, 2, 3, 4, 5, 6, 7, 8, 43 or 44 effective toinhibit the growth of multidrug resistant cells in combination with apharmaceutically acceptable carrier.
 53. The method of claim 52, furthercomprising administering an amount of a cytotoxic agent.
 54. The methodof claim 53, wherein the cytotoxic agent is an anticancer agent.
 55. Themethod of claim 54, wherein the anticancer agent is adriamycin.
 56. Themethod of claim 55, wherein the anticancer agent is vinblastin.
 57. Themethod of claim 55, wherein the anticancer agent is paclitaxel.
 58. Themethod of claim 55, wherein the effective amount of the compound isbetween about 0.01 mg/kg to about 25 mg/kg of body weight.